CONTRAST BETWEEN OSMIUM-FIXED AND PERMANGANATE-FIXED TOAD SPINAL GANGLIA

PubMed Central

Rosenbluth, Jack

1963-01-01

Chains of vesicles are prominent near the plasma membranes of both the neurons and satellite cells of osmium-fixed toad spinal ganglia. In permanganate-fixed specimens, however, such vesicles are absent, and in their place are continuous invaginations of the plasma membranes of these cells. The discrepancy suggests that the serried vesicles seen in osmium-fixed preparations arise through disintegration of plasma membrane invaginations, and do not represent active pinocytosis, as has been suggested previously. A second difference between ganglia fixed by these two methods is that rows of small, disconnected cytoplasmic globules occur in the sheaths of permanganate-fixed ganglia, but not in osmium-fixed samples. It is suggested that these globules arise from the breakdown of thin sheets of satellite cell cytoplasm which occur as continuous lamellae in osmium-fixed specimens. Possible mechanisms of these membrane reorganizations, and the relevance of these findings to other tissues, are discussed. PMID:13990905

Three-Dimensional Imaging of the Mouse Organ of Corti Cytoarchitecture for Mechanical Modeling

NASA Astrophysics Data System (ADS)

Puria, Sunil; Hartman, Byron; Kim, Jichul; Oghalai, John S.; Ricci, Anthony J.; Liberman, M. Charles

2011-11-01

Cochlear models typically use continuous anatomical descriptions and homogenized parameters based on two-dimensional images for describing the organ of Corti. To produce refined models based more closely on the actual cochlear cytoarchitecture, three-dimensional morphometric parameters of key mechanical structures are required. Towards this goal, we developed and compared three different imaging methods: (1) A fixed cochlear whole-mount preparation using the fluorescent dye Cellmask®, which is a molecule taken up by cell membranes and clearly delineates Deiters' cells, outer hair cells, and the phalangeal process, imaged using confocal microscopy; (2) An in situ fixed preparation with hair cells labeled using anti-prestin and supporting structures labeled using phalloidin, imaged using two-photon microscopy; and (3) A membrane-tomato (mT) mouse with fluorescent proteins expressed in all cell membranes, which enables two-photon imaging of an in situ live preparation with excellent visualization of the organ of Corti. Morphometric parameters including lengths, diameters, and angles, were extracted from 3D cellular surface reconstructions of the resulting images. Preliminary results indicate that the length of the phalangeal processes decreases from the first (inner most) to third (outer most) row of outer hair cells, and that their length also likely varies from base to apex and across species.

Exploration of mechanisms underlying the strain-rate-dependent mechanical property of single chondrocytes

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

Nguyen, Trung Dung; Gu, YuanTong, E-mail: yuantong.gu@qut.edu.au

2014-05-05

Based on the characterization by Atomic Force Microscopy, we report that the mechanical property of single chondrocytes has dependency on the strain-rates. By comparing the mechanical deformation responses and the Young's moduli of living and fixed chondrocytes at four different strain-rates, we explore the deformation mechanisms underlying this dependency property. We found that the strain-rate-dependent mechanical property of living cells is governed by both of the cellular cytoskeleton and the intracellular fluid when the fixed chondrocytes are mainly governed by their intracellular fluid, which is called the consolidation-dependent deformation behavior. Finally, we report that the porohyperelastic constitutive material model whichmore » can capture the consolidation-dependent behavior of both living and fixed chondrocytes is a potential candidature to study living cell biomechanics.« less

Gardnerella vaginalis and Lactobacillus sp in liquid-based cervical samples in healthy and disturbed vaginal flora using cultivation-independent methods.

PubMed

Klomp, Johanna M; Verbruggen, Banut-Sabine M; Korporaal, Hans; Boon, Mathilde E; de Jong, Pauline; Kramer, Gerco C; van Haaften, Maarten; Heintz, A Peter M

2008-05-01

Our objective was to determine the morphotype of the adherent bacteria in liquid-based cytology (LBC) in smears with healthy and disturbed vaginal flora. And to use PCR technology on the same fixed cell sample to establish DNA patterns of the 16S RNA genes of the bacteria in the sample. Thirty samples were randomly selected from a large group of cervical cell samples suspended in a commercial coagulant fixative "(BoonFix)." PCR was used to amplify DNA of five bacterial species: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, and Mycoplasma hominis. The LBC slides were then analyzed by light microscopy to estimate bacterial adhesion. DNA of lactobacilli was detected in all cell samples. Seventeen smears showed colonization with Gardnerella vaginalis (range 2.6 x 10(2)-3.0 x 10(5) bacteria/mul BoonFix sample). Two cases were identified as dysbacteriotic with high DNA values for Gardnerella vaginalis and low values for Lactobacillus crispatus. The sample with the highest concentration for Gardnerella vaginalis showed an unequivocal Gardnerella infection. This study indicates that the adherence pattern of a disturbed flora in liquid-based cervical samples can be identified unequivocally, and that these samples are suitable for quantitative PCR analysis. This cultivation independent method reveals a strong inverse relationship between Gardnerella vaginalis and Lactobacillus crispatus in dysbacteriosis and unequivocal Gardnerella infection.

Superficial Collagen Fibril Modulus and Pericellular Fixed Charge Density Modulate Chondrocyte Volumetric Behaviour in Early Osteoarthritis

PubMed Central

Turunen, Siru M.; Han, Sang Kuy; Herzog, Walter; Korhonen, Rami K.

2013-01-01

The aim of this study was to investigate if the experimentally detected altered chondrocyte volumetric behavior in early osteoarthritis can be explained by changes in the extracellular and pericellular matrix properties of cartilage. Based on our own experimental tests and the literature, the structural and mechanical parameters for normal and osteoarthritic cartilage were implemented into a multiscale fibril-reinforced poroelastic swelling model. Model simulations were compared with experimentally observed cell volume changes in mechanically loaded cartilage, obtained from anterior cruciate ligament transected rabbit knees. We found that the cell volume increased by 7% in the osteoarthritic cartilage model following mechanical loading of the tissue. In contrast, the cell volume decreased by 4% in normal cartilage model. These findings were consistent with the experimental results. Increased local transversal tissue strain due to the reduced collagen fibril stiffness accompanied with the reduced fixed charge density of the pericellular matrix could increase the cell volume up to 12%. These findings suggest that the increase in the cell volume in mechanically loaded osteoarthritic cartilage is primarily explained by the reduction in the pericellular fixed charge density, while the superficial collagen fibril stiffness is suggested to contribute secondarily to the cell volume behavior. PMID:23634175

Simultaneous Measurement of Multiple Mechanical Properties of Single Cells Using AFM by Indentation and Vibration.

PubMed

Zhang, Chuang; Shi, Jialin; Wang, Wenxue; Xi, Ning; Wang, Yuechao; Liu, Lianqing

2017-12-01

The mechanical properties of cells, which are the main characteristics determining their physical performance and physiological functions, have been actively studied in the fields of cytobiology and biomedical engineering and for the development of medicines. In this study, an indentation-vibration-based method is proposed to simultaneously measure the mechanical properties of cells in situ, including cellular mass (m), elasticity (k), and viscosity (c). The proposed measurement method is implemented based on the principle of forced vibration stimulated by simple harmonic force using an atomic force microscope (AFM) system integrated with a piezoelectric transducer as the substrate vibrator. The corresponding theoretical model containing the three mechanical properties is derived and used to perform simulations and calculations. Living and fixed human embryonic kidney 293 (HEK 293) cells were subjected to indentation and vibration to measure and compare their mechanical parameters and verify the proposed approach. The results that the fixed sample cells are more viscous and elastic than the living sample cells and the measured mechanical properties of cell are consistent within, but not outside of the central region of the cell, are in accordance with the previous studies. This work provides an approach to simultaneous measurement of the multiple mechanical properties of single cells using an integrated AFM system based on the principle force vibration and thickness-corrected Hertz model. This study should contribute to progress in biomedical engineering, cytobiology, medicine, early diagnosis, specific therapy and cell-powered robots.

Chromatid Paints: A New Method for Detecting Tumor-Specific Chromosomal Inversions

DTIC Science & Technology

1999-10-01

chromosomal DNA as a template for DNA polymerization. The cloning procedure requires copying DNA from fixed cells attached to a glass substrate. Any...achieved by initially fixing cells in methanol and adding acetic acid just before dropping cells onto coverslips. The procedure itself is a novel and...human hybrid cells containing one human #11 chromosome were fixed and dropped onto microscope coverslips. These cells had been synchronized by mitotic

Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

PubMed

Omairi-Nasser, Amin; Mariscal, Vicente; Austin, Jotham R; Haselkorn, Robert

2015-08-11

The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

NASA Astrophysics Data System (ADS)

Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

Cell fixation and preservation for droplet-based single-cell transcriptomics.

PubMed

Alles, Jonathan; Karaiskos, Nikos; Praktiknjo, Samantha D; Grosswendt, Stefanie; Wahle, Philipp; Ruffault, Pierre-Louis; Ayoub, Salah; Schreyer, Luisa; Boltengagen, Anastasiya; Birchmeier, Carmen; Zinzen, Robert; Kocks, Christine; Rajewsky, Nikolaus

2017-05-19

Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. By using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

PubMed

Görlitz, Frederik; Kelly, Douglas J; Warren, Sean C; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J; Stuhmeier, Frank; Neil, Mark A A; Tate, Edward W; Dunsby, Christopher; French, Paul M W

2017-01-18

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

PubMed Central

Warren, Sean C.; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A.; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Tate, Edward W.; Dunsby, Christopher; French, Paul M. W.

2017-01-01

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. PMID:28190060

An ethanol-based fixation method for anatomical and micro-morphological characterization of leaves of various tree species.

PubMed

Chieco, C; Rotondi, A; Morrone, L; Rapparini, F; Baraldi, R

2013-02-01

The use of formalin constitutes serious health hazards for laboratory workers. We investigated the suitability and performance of the ethanol-based fixative, FineFIX, as a substitute for formalin for anatomical and cellular structure investigations of leaves by light microscopy and for leaf surface and ultrastructural analysis by scanning electron microscopy (SEM). We compared the anatomical features of leaf materials prepared using conventional formalin fixation with the FineFIX. Leaves were collected from ornamental tree species commonly used in urban areas. FineFIX was also compared with glutaraldehyde fixation and air drying normally used for scanning electron microscopy to develop a new method for evaluating leaf morphology and microstructure in three ornamental tree species. The cytological features of the samples processed for histological analysis were well preserved by both fixatives as demonstrated by the absence of nuclear swelling or shrinkage, cell wall detachment or tissue flaking, and good presentation of cytoplasmic vacuolization. In addition, good preservation of surface details and the absence of shrinkage artefacts confirmed the efficacy of FineFIX fixation for SEM analysis. Cuticular wax was preserved only in air dried samples. Samples treated with chemical substances during the fixation and dehydration phases showed various alterations of the wax structures. In some air dried samples a loss of turgidity of the cells was observed that caused general wrinkling of the epidermal surfaces. Commercial FineFIX is an adequate substitute for formalin in histology and it can be applied successfully also for SEM investigation, while reducing the health risks of glutaraldehyde or other toxic fixatives. To investigate the potential for plants to absorb and capture particulates in air, which requires preservation of the natural morphology of trichomes and epicuticular waxes, a combination of FineFIX fixation and air drying is recommended.

Density gradient electrophoresis of cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

1985-01-01

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

Competitive clonal hematopoiesis in mouse chimeras explained by a stochastic model of stem cell organization.

PubMed

Roeder, Ingo; Kamminga, Leonie M; Braesel, Katrin; Dontje, Bert; de Haan, Gerald; Loeffler, Markus

2005-01-15

Many current experimental results show the necessity of new conceptual approaches to understand hematopoietic stem cell organization. Recently, we proposed a novel theoretical concept and a corresponding quantitative model based on microenvironment-dependent stem cell plasticity. The objective of our present work is to subject this model to an experimental test for the situation of chimeric hematopoiesis. Investigating clonal competition processes in DBA/2-C57BL/6 mouse chimeras, we observed biphasic chimerism development with initially increasing but long-term declining DBA/2 contribution. These experimental results were used to select the parameters of the mathematical model. To validate the model beyond this specific situation, we fixed the obtained parameter configuration to simulate further experimental settings comprising variations of transplanted DBA/2-C57BL/6 proportions, secondary transplantations, and perturbation of stabilized chimeras by cytokine and cytotoxic treatment. We show that the proposed model is able to consistently describe the situation of chimeric hematopoiesis. Our results strongly support the view that the relative growth advantage of strain-specific stem cells is not a fixed cellular property but is sensitively dependent on the actual state of the entire system. We conclude that hematopoietic stem cell organization should be understood as a flexible, self-organized rather than a fixed, preprogrammed process.

Mechanical Forces Program the Orientation of Cell Division during Airway Tube Morphogenesis.

PubMed

Tang, Zan; Hu, Yucheng; Wang, Zheng; Jiang, Kewu; Zhan, Cheng; Marshall, Wallace F; Tang, Nan

2018-02-05

Oriented cell division plays a key role in controlling organogenesis. The mechanisms for regulating division orientation at the whole-organ level are only starting to become understood. By combining 3D time-lapse imaging, mouse genetics, and mathematical modeling, we find that global orientation of cell division is the result of a combination of two types of spindles with distinct spindle dynamic behaviors in the developing airway epithelium. Fixed spindles follow the classic long-axis rule and establish their division orientation before metaphase. In contrast, rotating spindles do not strictly follow the long-axis rule and determine their division orientation during metaphase. By using both a cell-based mechanical model and stretching-lung-explant experiments, we showed that mechanical force can function as a regulatory signal in maintaining the stable ratio between fixed spindles and rotating spindles. Our findings demonstrate that mechanical forces, cell geometry, and oriented cell division function together in a highly coordinated manner to ensure normal airway tube morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

PubMed

Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

2016-08-01

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

PubMed

Li, Duo; Qiao, Zhenzhen; Yu, Yanru; Tang, Jinlu; He, Xiaoxiao; Shi, Hui; Ye, Xiaosheng; Lei, Yanli; Wang, Kemin

2018-01-25

A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.

The Use of Raman Tweezers and Chemometric Analysis to Discriminate the Urological Cell Lines, PC-3, LNCaP, BPH and MGH-U1

NASA Astrophysics Data System (ADS)

Harvey, T. J.; Hughes, C.; Ward, A. D.; Gazi, E.; Faria, E. Correia; Clarke, N. W.; Brown, M.; Snook, R.; Gardner, P.

2008-11-01

Here we report on investigations into using Raman optical tweezers to analyse both live and chemically fixed prostate and bladder cells. Spectra were subjected to chemometric analysis to discriminate and classify the cell types based on their spectra. Subsequent results revealed the potential of Raman tweezers as a potential clinical diagnostic tool.

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.

PubMed

Schläfli, A M; Berezowska, S; Adams, O; Langer, R; Tschan, M P

2015-05-05

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

PubMed

Laget, Sophie; Broncy, Lucile; Hormigos, Katia; Dhingra, Dalia M; BenMohamed, Fatima; Capiod, Thierry; Osteras, Magne; Farinelli, Laurent; Jackson, Stephen; Paterlini-Bréchot, Patrizia

2017-01-01

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

PubMed

Yaremko, M L; Kelemen, P R; Kutza, C; Barker, D; Westbrook, C A

1996-01-01

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible.

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Effect of Impurities on the Freezing Point of Zinc

NASA Astrophysics Data System (ADS)

Sun, Jianping; Rudtsch, Steffen; Niu, Yalu; Zhang, Lin; Wang, Wei; Den, Xiaolong

2017-03-01

The knowledge of the liquidus slope of impurities in fixed-point metal defined by the International Temperature Scale of 1990 is important for the estimation of uncertainties and correction of fixed point with the sum of individual estimates method. Great attentions are paid to the effect of ultra-trace impurities on the freezing point of zinc in the National Institute of Metrology. In the present work, the liquidus slopes of Ga-Zn, Ge-Zn were measured with the slim fixed-point cell developed through the doping experiments, and the temperature characteristics of the phase diagram of Fe-Zn were furthermore investigated. A quasi-adiabatic Zn fixed-point cell was developed with the thermometer well surrounded by the crucible with the pure metal, and the temperature uniformity of less than 20 mK in the region where the metal is located was obtained. The previous doping experiment of Pb-Zn with slim fixed-point cell was checked with quasi-adiabatic Zn fixed-point cell, and the result supports the previous liquidus slope measured with the traditional fixed-point realization.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

PubMed

Monzani, P S; Sangalli, J R; De Bem, T H C; Bressan, F F; Fantinato-Neto, P; Pimentel, J R V; Birgel-Junior, E H; Fontes, A M; Covas, D T; Meirelles, F V

2013-02-28

Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX.

PubMed

Cao, Jun; Shang, Chang-zhen; Lü, Li-hong; Qiu, De-chuan; Ren, Meng; Chen, Ya-jin; Min, Jun

2010-11-01

To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX. Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell-derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium. The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell-derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium. We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.

Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

PubMed

Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

2016-01-01

Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

Effect of pH on ion current through conical nanopores

NASA Astrophysics Data System (ADS)

Chander, M.; Kumar, R.; Kumar, S.; Kumar, N.

2018-05-01

Here, we examined ionic current behavior of conical nanopores at different pH and a fixed ion concentration of potassium halide (KCl). Conical shaped nanopores have been developed by chemical etching technique in polyethylene terephthalate (PET) membrane/foil of thickness 12 micron. For this we employed a self-assembled electrochemical cell having two chambers and the foil was fitted in the centre of cell. The nanopores were produced in the foil using etching and stopping solutions. The experimental results show that ionic current rectification (ICR) occurs through synthesized conical nanopores. Further, ion current increases significantly with increase of voltage from the base side of nanopores to the tip side at fixed pH of electrolyte.

Plant cell wall sugars: sweeteners for a bio-based economy.

PubMed

Van de Wouwer, Dorien; Boerjan, Wout; Vanholme, Bartel

2018-02-12

Global warming and the consequent climate change is one of the major environmental challenges we are facing today. The driving force behind the rise in temperature is our fossil-based economy, which releases massive amounts of the greenhouse gas carbon dioxide into the atmosphere. In order to reduce greenhouse gas emission, we need to scale down our dependency on fossil resources, implying that we need other sources for energy and chemicals to feed our economy. Here, plants have an important role to play; by means of photosynthesis, plants capture solar energy to split water and fix carbon derived from atmospheric carbon dioxide. A significant fraction of the fixed carbon ends up as polysaccharides in the plant cell wall. Fermentable sugars derived from cell wall polysaccharides form an ideal carbon source for the production of bio-platform molecules. However, a major limiting factor in the use of plant biomass as feedstock for the bio-based economy is the complexity of the plant cell wall and its recalcitrance towards deconstruction. To facilitate the release of fermentable sugars during downstream biomass processing, the composition and structure of the cell wall can be engineered. Different strategies to reduce cell wall recalcitrance will be described in this review. The ultimate goal is to obtain a tailor-made biomass, derived from plants with a cell wall optimized for particular industrial or agricultural applications, without affecting plant growth and development. This article is protected by copyright. All rights reserved.

Establishment of the Co-C Eutectic Fixed-Point Cell for Thermocouple Calibrations at NIMT

NASA Astrophysics Data System (ADS)

Ongrai, O.; Elliott, C. J.

2017-08-01

In 2015, NIMT first established a Co-C eutectic temperature reference (fixed-point) cell measurement capability for thermocouple calibration to support the requirements of Thailand's heavy industries and secondary laboratories. The Co-C eutectic fixed-point cell is a facility transferred from NPL, where the design was developed through European and UK national measurement system projects. In this paper, we describe the establishment of a Co-C eutectic fixed-point cell for thermocouple calibration at NIMT. This paper demonstrates achievement of the required furnace uniformity, the Co-C plateau realization and the comparison data between NIMT and NPL Co-C cells by using the same standard Pt/Pd thermocouple, demonstrating traceability. The NIMT measurement capability for noble metal type thermocouples at the new Co-C eutectic fixed point (1324.06°C) is estimated to be within ± 0.60 K (k=2). This meets the needs of Thailand's high-temperature thermocouple users—for which previously there has been no traceable calibration facility.

An efficient auto TPT stitch guidance generation for optimized standard cell design

NASA Astrophysics Data System (ADS)

Samboju, Nagaraj C.; Choi, Soo-Han; Arikati, Srini; Cilingir, Erdem

2015-03-01

As the technology continues to shrink below 14nm, triple patterning lithography (TPT) is a worthwhile lithography methodology for printing dense layers such as Metal1. However, this increases the complexity of standard cell design, as it is very difficult to develop a TPT compliant layout without compromising on the area. Hence, this emphasizes the importance to have an accurate stitch generation methodology to meet the standard cell area requirement as defined by the technology shrink factor. In this paper, we present an efficient auto TPT stitch guidance generation technique for optimized standard cell design. The basic idea here is to first identify the conflicting polygons based on the Fix Guidance [1] solution developed by Synopsys. Fix Guidance is a reduced sub-graph containing minimum set of edges along with the connecting polygons; by eliminating these edges in a design 3-color conflicts can be resolved. Once the conflicting polygons are identified using this method, they are categorized into four types [2] - (Type 1 to 4). The categorization is based on number of interactions a polygon has with the coloring links and the triangle loops of fix guidance. For each type a certain criteria for keep-out region is defined, based on which the final stitch guidance locations are generated. This technique provides various possible stitch locations to the user and helps the user to select the best stitch location considering both design flexibility (max. pin access/small area) and process-preferences. Based on this technique, a standard cell library for place and route (P and R) can be developed with colorless data and a stitch marker defined by designer using our proposed method. After P and R, the full chip (block) would contain the colorless data and standard cell stitch markers only. These stitch markers are considered as "must be stitch" candidates. Hence during full chip decomposition it is not required to generate and select the stitch markers again for the complete data; therefore, the proposed method reduces the decomposition time significantly.

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

PubMed Central

Yaremko, M. L.; Kelemen, P. R.; Kutza, C.; Barker, D.; Westbrook, C. A.

1996-01-01

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible. Images Figure 1 Figure 2 Figure 3 PMID:8546231

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

PubMed Central

Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

2009-01-01

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

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.

A micromanipulation cell including a tool changer

NASA Astrophysics Data System (ADS)

Clévy, Cédric; Hubert, Arnaud; Agnus, Joël; Chaillet, Nicolas

2005-10-01

This paper deals with the design, fabrication and characterization of a tool changer for micromanipulation cells. This tool changer is part of a manipulation cell including a three linear axes robot and a piezoelectric microgripper. All these parts are designed to perform micromanipulation tasks in confined spaces such as a microfactory or in the chamber of a scanning electron microscope (SEM). The tool changer principle is to fix a pair of tools (i.e. the gripper tips) either on the tips of the microgripper actuator (piezoceramic bulk) or on a tool magazine. The temperature control of a thermal glue enables one to fix or release this pair of tools. Liquefaction and solidification are generated by surface mounted device (SMD) resistances fixed on the surface of the actuator or magazine. Based on this principle, the tool changer can be adapted to other kinds of micromanipulation cells. Hundreds of automatic tool exchanges were performed with a maximum positioning error between two consecutive tool exchanges of 3.2 µm, 2.3 µm and 2.8 µm on the X, Y and Z axes respectively (Z refers to the vertical axis). Finally, temperature measurements achieved under atmospheric pressure and in a vacuum environment and pressure measurements confirm the possibility of using this device in the air as well as in a SEM.

Fluorescent probes for nucleic Acid visualization in fixed and live cells.

PubMed

Boutorine, Alexandre S; Novopashina, Darya S; Krasheninina, Olga A; Nozeret, Karine; Venyaminova, Alya G

2013-12-11

This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

PubMed Central

Laget, Sophie; Dhingra, Dalia M.; BenMohamed, Fatima; Capiod, Thierry; Osteras, Magne; Farinelli, Laurent; Jackson, Stephen; Paterlini-Bréchot, Patrizia

2017-01-01

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion. PMID:28060956

Free-zone electrophoresis of animal cells. 1: Experiments on cell-cell interactions

NASA Technical Reports Server (NTRS)

Todd, P. W.; Hjerten, S.

1985-01-01

The electrophoretically migrating zones wasa monitored. The absence of fluid flows in the direction of migration permits direct measurement of electrophoretic velocities of any material. Sedimentation is orthogonal to electrokinetic motion and the effects of particle-particle interaction on electrophoretic mobility is studied by free zone electrophoresis. Fixed erythrocytes at high concentrations, mixtures of fixed erythrocytes from different animal species, and mixtures of cultured human cells were studied in low ionic strength buffers. The electrophoretic velocity of fixed erythrocytes was not altered by increasing cell concentration or by the mixing of erythrocytes from different species. When zones containing cultured human glial cells and neuroblastoma cells are permitted to interact during electrophoresis, altered migration patterns occur. It is found that cell-cell interactions depends upon cell type.

Indirect determination of the thermodynamic temperature of the copper point by a multi-fixed-point technique

NASA Astrophysics Data System (ADS)

Battuello, M.; Florio, M.; Girard, F.

2010-06-01

An indirect determination of the thermodynamic temperature of the fixed point of copper was made at INRIM by measuring four cells with a Si-based and an InGaAs-based precision radiation thermometer carrying approximated thermodynamic scales realized up to the Ag point. An average value TCu = 1357.840 K was found with a standard uncertainty of 0.047 K. A consequent (T - T90)Cu value of 70 mK can be derived which is 18 mK higher than, but consistent with, the presently available (T - T90)Cu as elaborated by the CCT-WG4.

Development of Fixed-Point Cells at the SMU

NASA Astrophysics Data System (ADS)

Ďuriš, S.; Ranostaj, J.; Palenčár, R.

2008-06-01

One of the research programs realized at the thermometry laboratory of the Slovak Institute of Metrology (SMU) in recent years has focused on the development of fixed-point cells. In the frame of this research, several primary cells for realization of the International Temperature Scale of 1990 (ITS-90) and several secondary cells for industrial thermometer calibrations were built and studied. This article discusses primary cells for the gallium and mercury fixed points and miniature cells for the zinc point that were developed at the SMU. Information about the cell designs is provided, the materials that were used are specified, and the procedures for their manufacture are described. Briefly, the realization of the fixed points of mercury, gallium, and zinc by using these cells is also described. Many experiments were carried out to study the characteristics of these cells. One of the gallium cells was compared with the circulating transfer cell during the key comparison CCT-K3, and it and the mercury cell were used for the EUROMET Project No. 552. The results of the experiments together with the results of the comparisons show the high quality of these cells. Secondary zinc-point cells were compared against SMU primary zinc-point cells. The comparison shows agreement within 0.12 mK.

Construction of a Cr3C2-C Peritectic Point Cell for Thermocouple Calibration

NASA Astrophysics Data System (ADS)

Ogura, Hideki; Deuze, Thierry; Morice, Ronan; Ridoux, Pascal; Filtz, Jean-Remy

The melting points of Cr3C2-C peritectic (1826°C) and Cr7C3-Cr3C2 eutectic (1742°C) alloys as materials for high-temperature fixed point cells are investigated for the use of thermocouple calibration. Pretests are performed to establish a suitable procedure for constructing contact thermometry cells based on such chromium-carbon mixtures. Two cells are constructed following two different possible procedures. The above two melting points are successfully observed for one of these cells using tungsten-rhenium alloy thermocouples.

Ultrastructural and mechanical changes in tubular epithelial cells by angiotensin II and aldosterone as observed with atomic force microscopy.

PubMed

Quan, Fu-Shi; Jeong, Kyung Hwan; Lee, Gi-Ja

2018-07-01

Tubular epithelial cells (TECs) play an important pathophysiological role in the promotion of renal fibrosis. Quantitative analysis of the mechanical changes in TECs may be helpful in evaluating novel pharmacological strategies. Atomic force microscopy (AFM) is a common nanotechnology tool used for imaging and measuring interaction forces in biological systems. In this study, we used AFM to study ultrastructural and mechanical changes in TECs mediated by the renin-angiotensin-aldosterone system. We quantitatively analyzed changes in the mechanical properties of TECs using three extrinsic factors, namely, chemical fixation, angiotensin II (AT II), and aldosterone (AD). Fixed TECs were 11 times stiffer at the cell body and 3 times stiffer at the cell-cell junction compared to live TECs. After stimulation with AT II, live TECs were four times stiffer at the junctional area than at the cell body, while fixed TECs after AT II stimulation were approximately two times stiffer at the both cell body and cell-cell junction compared to fixed unstimulated TECs. Fixed TECs also reflected changes in the mechanical properties of TECs at the cell body region after AD stimulation. Together, our results suggest that cell stiffness at the cell body region may serve as an effective index for evaluating drugs and stimulation, regardless of whether the cells are live or fixed at the time of analysis. In addition, studying the changes to the intrinsic mechanical property of TECs after application of external stimuli may be useful for investigating pathophysiologic mechanisms and effective therapeutic strategies for renal injury. Copyright © 2018 Elsevier Ltd. All rights reserved.

Visualization of channels connecting cells in filamentous nitrogen-fixing cyanobacteria.

PubMed

Omairi-Nasser, Amin; Haselkorn, Robert; Austin, Jotham

2014-07-01

Cyanobacteria, formerly called blue-green algae, are abundant bacteria that carry out green plant photosynthesis, fixing CO2 and generating O2. Many species can also fix N2 when reduced nitrogen sources are scarce. Many studies imply the existence of intracellular communicating channels in filamentous cyanobacteria, in particular, the nitrogen-fixing species. In a species such as Anabaena, growth in nitrogen-depleted medium, in which ∼10% of the cells differentiate into anaerobic factories for nitrogen fixation (heterocysts), requires the transport of amino acids from heterocysts to vegetative cells, and reciprocally, the transport of sugar from vegetative cells to heterocysts. Convincing physical evidence for such channels has been slim. Using improved preservation of structure by high-pressure rapid freezing of samples for electron microscopy, coupled with high-resolution 3D tomography, it has been possible to visualize and measure the dimensions of channels that breach the peptidoglycan between vegetative cells and between heterocysts and vegetative cells. The channels appear to be straight tubes, 21 nm long and 14 nm in diameter for the latter and 12 nm long and 12 nm in diameter for the former.-Omairi-Nasser, A., Haselkorn, R., Austin, J. II. Visualization of channels connecting cells in filamentous nitrogen-fixing cyanobacteria. © FASEB.

Using oligonucleotide aptamer probes for immunostaining of formalin-fixed and paraffin-embedded tissues

PubMed Central

Zeng, Zihua; Zhang, Peng; Zhao, Nianxi; Sheehan, Andrea M; Tung, Ching-Hsuan; Chang, Chung-Che; Zu, Youli

2011-01-01

For tissue immunostaining, antibodies are currently the only clinically validated and commercially available probes. Aptamers, which belong to a class of small molecule ligands composed of short single-stranded oligonucleotides, have emerged as probes over the last several decades; however, their potential clinical value has not yet been fully explored. Using cultured cells and an RNA-based CD30 aptamer, we recently demonstrated that the synthetic aptamer is useful as a specific probe for flow cytometric detection of CD30-expressing lymphoma cells. In this study, we further validated the use of this aptamer probe for immunostaining of formalin-fixed and paraffin-embedded lymphoma tissues. Using CD30 antibody as a standard control, we demonstrated that the synthetic CD30 aptamer specifically recognized and immunostained tumor cells of classical Hodgkin lymphoma and anaplastic large cell lymphoma, but did not react with background cells within tumor sites. Notably, the CD30 aptamer probe optimally immunostained lymphoma cells with lower temperature antigen retrieval (37 vs 96°C for antibody) and shorter probing reaction times (20 vs 90 min for antibody) than typical antibody immunostaining protocols. In addition, the CD30 aptamer probe showed no nonspecific background staining of cell debris in necrotic tissue and exhibited no cross-reaction to tissues that do not express CD30, as confirmed by a standard CD30 antibody staining. Therefore, our findings indicate that the synthetic oligonucleotide CD30 aptamer can be used as a probe for immunostaining of fixed tissue sections for disease diagnosis. PMID:20693984

Investigation of the Parameters of Sealed Triple-Point Cells for Cryogenic Gases

NASA Astrophysics Data System (ADS)

Fellmuth, B.; Wolber, L.

2011-01-01

An overview of the parameters of a large number of sealed triple-point cells for the cryogenic gases hydrogen, oxygen, neon, and argon is given that have been determined within the framework of an international star intercomparison to optimize the measurement of melting curves as well as to establish complete and reliable uncertainty budgets for the realization of temperature fixed points. Special emphasis is given to the question, whether the parameters are primarily influenced by the cell design or the properties of the fixed-point samples. For explaining surprisingly large periods of the thermal recovery after the heat pulses of the intermittent heating through the melting range, a simple model is developed based on a newly defined heat-capacity equivalent, which considers the heat of fusion and a melting-temperature inhomogeneity. The analysis of the recovery using a graded set of exponential functions containing different time constants is also explained in detail.

Modulating the fixed charge density in silicon nitride films while monitoring the surface recombination velocity by photoluminescence imaging

NASA Astrophysics Data System (ADS)

Bazilchuk, Molly; Haug, Halvard; Marstein, Erik Stensrud

2015-04-01

Several important semiconductor devices such as solar cells and photodetectors may be fabricated based on surface inversion layer junctions induced by fixed charge in a dielectric layer. Inversion layer junctions can easily be fabricated by depositing layers with a high density of fixed charge on a semiconducting substrate. Increasing the fixed charge improves such devices; for instance, the efficiency of a solar cell can be substantially increased by reducing the surface recombination velocity, which is a function of the fixed charge density. Methods for increasing the charge density are therefore of interest. In this work, the fixed charge density in silicon nitride layers deposited by plasma enhanced chemical vapor deposition is increased to very high values above 1 × 1013 cm-2 after the application of an external voltage to a gate electrode. The effect of the fixed charge density on the surface recombination velocity was experimentally observed using the combination of capacitance-voltage characterization and photoluminescence imaging, showing a significant reduction in the surface recombination velocity for increasing charge density. The surface recombination velocity vs. charge density data was analyzed using a numerical device model, which indicated the presence of a sub-surface damage region formed during deposition of the layers. Finally, we have demonstrated that the aluminum electrodes used for charge injection may be chemically removed in phosphoric acid without loss of the underlying charge. The injected charge was shown to be stable for a prolonged time period, leading us to propose charge injection in silicon nitride films by application of soaking voltage as a viable method for fabricating inversion layer devices.

A Comparative Experimental Study of Fixed Temperature and Fixed Heat Flux Boundary Conditions in Turbulent Thermal Convection

NASA Astrophysics Data System (ADS)

Huang, Shi-Di; Wang, Fei; Xi, Heng-Dong; Xia, Ke-Qing

2014-11-01

We report an experimental study of the influences of thermal boundary condition in turbulent thermal convection. Two configurations were examined: one was fixed heat flux at the bottom boundary and fixed temperature at the top (HC cells); the other was fixed temperature at both boundaries (CC cells). It is found that the flow strength in the CC cells is on average 9% larger than that in the HC ones, which could be understood as change in plume emission ability under different boundary conditions. It is further found, rather surprisingly, that flow reversals of the large-scale circulation occur more frequently in the CC cell, despite a stronger large-scale flow and more uniform temperature distribution over the boundaries. These findings provide new insights into turbulent thermal convection and should stimulate further studies, especially experimental ones. This work is supported by the Hong Kong Research Grants Council under Grant No. CUHK 403712.

Model-based approach for design verification and co-optimization of catastrophic and parametric-related defects due to systematic manufacturing variations

NASA Astrophysics Data System (ADS)

Perry, Dan; Nakamoto, Mark; Verghese, Nishath; Hurat, Philippe; Rouse, Rich

2007-03-01

Model-based hotspot detection and silicon-aware parametric analysis help designers optimize their chips for yield, area and performance without the high cost of applying foundries' recommended design rules. This set of DFM/ recommended rules is primarily litho-driven, but cannot guarantee a manufacturable design without imposing overly restrictive design requirements. This rule-based methodology of making design decisions based on idealized polygons that no longer represent what is on silicon needs to be replaced. Using model-based simulation of the lithography, OPC, RET and etch effects, followed by electrical evaluation of the resulting shapes, leads to a more realistic and accurate analysis. This analysis can be used to evaluate intelligent design trade-offs and identify potential failures due to systematic manufacturing defects during the design phase. The successful DFM design methodology consists of three parts: 1. Achieve a more aggressive layout through limited usage of litho-related recommended design rules. A 10% to 15% area reduction is achieved by using more aggressive design rules. DFM/recommended design rules are used only if there is no impact on cell size. 2. Identify and fix hotspots using a model-based layout printability checker. Model-based litho and etch simulation are done at the cell level to identify hotspots. Violations of recommended rules may cause additional hotspots, which are then fixed. The resulting design is ready for step 3. 3. Improve timing accuracy with a process-aware parametric analysis tool for transistors and interconnect. Contours of diffusion, poly and metal layers are used for parametric analysis. In this paper, we show the results of this physical and electrical DFM methodology at Qualcomm. We describe how Qualcomm was able to develop more aggressive cell designs that yielded a 10% to 15% area reduction using this methodology. Model-based shape simulation was employed during library development to validate architecture choices and to optimize cell layout. At the physical verification stage, the shape simulator was run at full-chip level to identify and fix residual hotspots on interconnect layers, on poly or metal 1 due to interaction between adjacent cells, or on metal 1 due to interaction between routing (via and via cover) and cell geometry. To determine an appropriate electrical DFM solution, Qualcomm developed an experiment to examine various electrical effects. After reporting the silicon results of this experiment, which showed sizeable delay variations due to lithography-related systematic effects, we also explain how contours of diffusion, poly and metal can be used for silicon-aware parametric analysis of transistors and interconnect at the cell-, block- and chip-level.

Early Detection of Breast Cancer Using Molecular Beacons

DTIC Science & Technology

2008-01-01

a molecular beacon (MB)-based approach for direct examination of gene expression in viable and fixed cells (2, 3). This objective of proposed study ...can be distinguished from normal cells (dark) (Figure 1) (2, 3, 8). Recently, a class of new fluorescent emitting nanoparticles, semiconductor ...morphological classification. This method may offer a simple and fast procedure to detect biomarker gene expression in clinical samples. Our study results

A rapid, automated approach for quantitation of rotavirus and reovirus infectivity.

PubMed

Iskarpatyoti, Jason A; Willis, Janet Z; Guan, John; Morse, E Ashley; Ikizler, Miné; Wetzel, J Denise; Dermody, Terence S; Contractor, Nikhat

2012-09-01

Current microscopy-based approaches for immunofluorescence detection of viral infectivity are time consuming and labor intensive and can yield variable results subject to observer bias. To circumvent these problems, we developed a rapid and automated infrared immunofluorescence imager-based infectivity assay for both rotavirus and reovirus that can be used to quantify viral infectivity and infectivity inhibition. For rotavirus, monolayers of MA104 cells were infected with simian strain SA-11 or SA-11 preincubated with rotavirus-specific human IgA. For reovirus, monolayers of either HeLa S3 cells or L929 cells were infected with strains type 1 Lang (T1L), type 3 Dearing (T3D), or either virus preincubated with a serotype-specific neutralizing monoclonal antibody (mAb). Infected cells were fixed and incubated with virus-specific polyclonal antiserum, followed by an infrared fluorescence-conjugated secondary antibody. Well-to-well variation in cell number was normalized using fluorescent reagents that stain fixed cells. Virus-infected cells were detected by scanning plates using an infrared imager, and results were obtained as a percent response of fluorescence intensity relative to a virus-specific standard. An expected dose-dependent inhibition of both SA-11 infectivity with rotavirus-specific human IgA and reovirus infectivity with T1L-specific mAb 5C6 and T3D-specific mAb 9BG5 was observed, confirming the utility of this assay for quantification of viral infectivity and infectivity blockade. The imager-based viral infectivity assay fully automates data collection and provides an important advance in technology for applications such as screening for novel modulators of viral infectivity. This basic platform can be adapted for use with multiple viruses and cell types. Copyright © 2012 Elsevier B.V. All rights reserved.

Fixed-Cell Imaging of Schizosaccharomyces pombe.

PubMed

Hagan, Iain M; Bagley, Steven

2016-07-01

The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. © 2016 Cold Spring Harbor Laboratory Press.

Improved Method for Linear B-Cell Epitope Prediction Using Antigen’s Primary Sequence

PubMed Central

Raghava, Gajendra P. S.

2013-01-01

One of the major challenges in designing a peptide-based vaccine is the identification of antigenic regions in an antigen that can stimulate B-cell’s response, also called B-cell epitopes. In the past, several methods have been developed for the prediction of conformational and linear (or continuous) B-cell epitopes. However, the existing methods for predicting linear B-cell epitopes are far from perfection. In this study, an attempt has been made to develop an improved method for predicting linear B-cell epitopes. We have retrieved experimentally validated B-cell epitopes as well as non B-cell epitopes from Immune Epitope Database and derived two types of datasets called Lbtope_Variable and Lbtope_Fixed length datasets. The Lbtope_Variable dataset contains 14876 B-cell epitope and 23321 non-epitopes of variable length where as Lbtope_Fixed length dataset contains 12063 B-cell epitopes and 20589 non-epitopes of fixed length. We also evaluated the performance of models on above datasets after removing highly identical peptides from the datasets. In addition, we have derived third dataset Lbtope_Confirm having 1042 epitopes and 1795 non-epitopes where each epitope or non-epitope has been experimentally validated in at least two studies. A number of models have been developed to discriminate epitopes and non-epitopes using different machine-learning techniques like Support Vector Machine, and K-Nearest Neighbor. We achieved accuracy from ∼54% to 86% using diverse s features like binary profile, dipeptide composition, AAP (amino acid pair) profile. In this study, for the first time experimentally validated non B-cell epitopes have been used for developing method for predicting linear B-cell epitopes. In previous studies, random peptides have been used as non B-cell epitopes. In order to provide service to scientific community, a web server LBtope has been developed for predicting and designing B-cell epitopes (http://crdd.osdd.net/raghava/lbtope/). PMID:23667458

Senescent changes in the ribosomes of animal cells in vivo and in vitro

NASA Technical Reports Server (NTRS)

Miquel, J.; Johnson, J. E., Jr.

1979-01-01

The paper examines RNA-ribosomal changes observed in protozoa and fixed postmitotic cells, as well as the characteristics of intermitotic cells. Attention is given to a discussion of the implications of the reported ribosomal changes as to the senescent deterioration of protein synthesis and physiological functions. A survey of the literature suggests that, while the data on ribosomal change in dividing cells both in vivo and in vitro are inconclusive, there is strong histological and biochemical evidence in favor of some degree of quantitative ribosomal loss in fixed postmitotic cells. Since these decreases in ribosomes are demonstrated in differential cells from nematodes, insects and mammals, they may represent a universal manifestation of cytoplasmic senescence in certain types of fixed postmitotic animal cells. The observed variability in ribosomal loss for cells belonging to the same type suggests that this involution phenomenon is rather related to the wear and tear suffered by a particular cell.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

PubMed

Carriel, Víctor; Campos, Antonio; Alaminos, Miguel; Raimondo, Stefania; Geuna, Stefano

2017-01-01

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

Comparison of corneal endothelial image analysis by Konan SP8000 noncontact and Bio-Optics Bambi systems.

PubMed

Benetz, B A; Diaconu, E; Bowlin, S J; Oak, S S; Laing, R A; Lass, J H

1999-01-01

Compare corneal endothelial image analysis by Konan SP8000 and Bio-Optics Bambi image-analysis systems. Corneal endothelial images from 98 individuals (191 eyes), ranging in age from 4 to 87 years, with a normal slit-lamp examination and no history of ocular trauma, intraocular surgery, or intraocular inflammation were obtained by the Konan SP8000 noncontact specular microscope. One observer analyzed these images by using the Konan system and a second observer by using the Bio-Optics Bambi system. Three methods of analyses were used: a fixed-frame method to obtain cell density (for both Konan and Bio-Optics Bambi) and a "dot" (Konan) or "corners" (Bio-Optics Bambi) method to determine morphometric parameters. The cell density determined by the Konan fixed-frame method was significantly higher (157 cells/mm2) than the Bio-Optics Bambi fixed-frame method determination (p<0.0001). However, the difference in cell density, although still statistically significant, was smaller and reversed comparing the Konan fixed-frame method with both Konan dot and Bio-Optics Bambi comers method (-74 cells/mm2, p<0.0001; -55 cells/mm2, p<0.0001, respectively). Small but statistically significant morphometric analyses differences between Konan and Bio-Optics Bambi were seen: cell density, +19 cells/mm2 (p = 0.03); cell area, -3.0 microm2 (p = 0.008); and coefficient of variation, +1.0 (p = 0.003). There was no statistically significant difference between these two methods in the percentage of six-sided cells detected (p = 0.55). Cell densities measured by the Konan fixed-frame method were comparable with Konan and Bio-Optics Bambi's morphometric analysis, but not with the Bio-Optics Bambi fixed-frame method. The two morphometric analyses were comparable with minimal or no differences for the parameters that were studied. The Konan SP8000 endothelial image-analysis system may be useful for large-scale clinical trials determining cell loss; its noncontact system has many clinical benefits (including patient comfort, safety, ease of use, and short procedure time) and provides reliable cell-density calculations.

Small copper fixed-point cells of the hybrid type to be used in place of normal larger cells

NASA Astrophysics Data System (ADS)

Battuello, M.; Girard, F.; Florio, M.

2012-10-01

Two small cells for the realization of the fixed point of copper were constructed and investigated at INRIM. They are of the same hybrid design generally adopted for the eutectic high-temperature fixed-point cells, namely a structure with a sacrificial graphite sleeve and a layer of flexible carbon-carbon composite sheet (C/C sheet). Because of the largely different design with respect to the cells normally adopted for the construction of pure metal fixed points, they were compared and characterized with respect to the normal cells used at INRIM for the ITS-90 realization. Two different furnaces were used to compare hybrid and normal cells. One of the hybrid cells was also used in different configurations, i.e. without the C/C sheet and with two layers of sheet. The cells were compared with different operative conditions, i.e. temperature settings of the furnaces for inducing the freeze, and repeatability and reproducibility were investigated. Freezing temperature and shape of the plateaux obtained under the different conditions were analysed. As expected the duration of the plateaux obtained with the hybrid cells is considerably shorter than with the normal cell, but this does not affect the results in terms of freezing temperature. Measurements with the modified cell showed that the use of a double C/C sheet may improve both repeatability and reproducibility of the plateaux.

Inhibition of nitrogen-fixing activity of the cyanobiont affects the localization of glutamine synthetase in hair cells of Azolla.

PubMed

Uheda, Eiji; Maejima, Kazuhiro

2009-10-15

In the Azolla-Anabaena association, the host plant Azolla efficiently incorporates and assimilates ammonium ions that are released from the nitrogen-fixing cyanobiont, probably via glutamine synthetase (GS; EC 6.3.1.2) in hair cells, which are specialized cells protruding into the leaf cavity. In order to clarify the regulatory mechanism underlying ammonium assimilation in the Azolla-Anabaena association, Azolla plants were grown under an argon environment (Ar), in which the nitrogen-fixing activity of the cyanobiont was inhibited specifically and completely. The localization of GS in hair cells was determined by immunoelectron microscopy and quantitative analysis of immunogold labeling. Azolla plants grew healthily under Ar when nitrogen sources, such as NO(3)(-) and NH(4)(+), were provided in the growth medium. Both the number of cyanobacterial cells per leaf and the heterocyst frequency of the plants under Ar were similar to those of plants in a nitrogen environment (N(2)). In hair cells of plants grown under Ar, regardless of the type of nitrogen source provided, only weak labeling of GS was observed in the cytoplasm and in chloroplasts. In contrast, in hair cells of plants grown under N(2), abundant labeling of GS was observed in both sites. These findings indicate that specific inhibition of the nitrogen-fixing activity of the cyanobiont affects the localization of GS isoenzymes. Ammonium fixed and released by the cyanobiont could stimulate GS synthesis in hair cells. Simultaneously, the abundant GS, probably GS1, in these cells, could assimilate ammonium rapidly.

Dose Rationalization of Pembrolizumab and Nivolumab Using Pharmacokinetic Modeling and Simulation and Cost Analysis.

PubMed

Ogungbenro, Kayode; Patel, Alkesh; Duncombe, Robert; Nuttall, Richard; Clark, James; Lorigan, Paul

2018-04-01

Pembrolizumab and nivolumab are highly selective anti-programmed cell death 1 (PD-1) antibodies approved for the treatment of advanced malignancies. Variable exposure and significant wastage have been associated with body size dosing of monoclonal antibodies (mAbs). The following dosing strategies were evaluated using simulations: body weight, dose banding, fixed dose, and pharmacokinetic (PK)-based methods. The relative cost to body weight dosing for band, fixed 150 mg and 200 mg, and PK-derived strategies were -15%, -25%, + 7%, and -16% for pembrolizumab and -8%, -6%, and -10% for band, fixed, and PK-derived strategies for nivolumab, respectively. Relative to mg/kg doses, the median exposures were -1.0%, -4.6%, + 27.1%, and +3.0% for band, fixed 150 mg, fixed 200 mg, and PK-derived strategies, respectively, for pembrolizumab and -3.1%, + 1.9%, and +1.4% for band, fixed 240 mg, and PK-derived strategies, respectively, for nivolumab. Significant wastage can be reduced by alternative dosing strategies without compromising exposure and efficacy. © 2017 American Society for Clinical Pharmacology and Therapeutics.

Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle.

PubMed

Wajih, Nadeem; Hutson, Susan M; Owen, John; Wallin, Reidar

2005-09-09

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.

Effects of formalin fixation on tissue optical properties of in-vitro brain samples

NASA Astrophysics Data System (ADS)

Anand, Suresh; Cicchi, Riccardo; Martelli, Fabrizio; Giordano, Flavio; Buccoliero, Anna Maria; Guerrini, Renzo; Pavone, Francesco S.

2015-03-01

Application of light spectroscopy based techniques for the detection of cancers have emerged as a promising approach for tumor diagnostics. In-vivo or freshly excised samples are normally used for point spectroscopic studies. However, ethical issues related to in-vivo studies, rapid decay of surgically excised tissues and sample availability puts a limitation on in-vivo and in-vitro studies. There has been a few studies reported on the application of formalin fixed samples with good discrimination capability. Usually formalin fixation is performed to prevent degradation of tissues after surgical resection. Fixing tissues in formalin prevents cell death by forming cross-linkages with proteins. Previous investigations have revealed that washing tissues fixed in formalin using phosphate buffered saline is known to reduce the effects of formalin during spectroscopic measurements. But this could not be the case with reflectance measurements. Hemoglobin is a principal absorbing medium in biological tissues in the visible range. Formalin fixation causes hemoglobin to seep out from red blood cells. Also, there could be alterations in the refractive index of tissues when fixed in formalin. In this study, we propose to investigate the changes in tissue optical properties between freshly excised and formalin fixed brain tissues. The results indicate a complete change in the spectral profile in the visible range where hemoglobin has its maximum absorption peaks. The characteristic bands of oxy-hemoglobin at 540, 580 nm and deoxy-hemoglobin at 555 nm disappear in the case of samples fixed in formalin. In addition, an increased spectral intensity was observed for the wavelengths greater than 650 nm where scattering phenomena are presumed to dominate.

General Staining and Segmentation Procedures for High Content Imaging and Analysis.

PubMed

Chambers, Kevin M; Mandavilli, Bhaskar S; Dolman, Nick J; Janes, Michael S

2018-01-01

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

Studies on the Ionic Permeability of Muscle Cells and their Models

PubMed Central

Ling, Gilbert N.; Ochsenfeld, Margaret M.

1965-01-01

We studied the effect an alkali-metal ion exercised on the rate of entry of another alkali-metal ion into frog sartorius muscle cells and their models (i.e., ion exchange resin and sheep's wool). In the case of frog muscle, it was shown that the interaction fell into one of four categories; competition, facilitation, and two types of indifference. The observed pK value (4.6 to 4.7) of the surface anionic groups that combine with the alkali-metal ions suggests that they are β- or γ-carboxyl groups of proteins on the cell surface. The results were compared with four theoretical models which included three membrane models (continuous lipoid membrane with carrier; leaky membrane with carrier; membrane with fixed ionic sites) and one bulk-phase model. This comparison led to the conclusion that the only model that is self-consistent and agrees with all of the experimental facts is the one based on the concept that the entire living cell represents a proteinaceous fixed-charge system; this model correctly predicts all four types of interaction observed. PMID:5884012

Tools for visualization of phosphoinositides in the cell nucleus.

PubMed

Kalasova, Ilona; Fáberová, Veronika; Kalendová, Alžběta; Yildirim, Sukriye; Uličná, Lívia; Venit, Tomáš; Hozák, Pavel

2016-04-01

Phosphoinositides (PIs) are glycerol-based phospholipids containing hydrophilic inositol ring. The inositol ring is mono-, bis-, or tris-phosphorylated yielding seven PIs members. Ample evidence shows that PIs localize both to the cytoplasm and to the nucleus. However, tools for direct visualization of nuclear PIs are limited and many studies thus employ indirect approaches, such as staining of their metabolic enzymes. Since localization and mobility of PIs differ from their metabolic enzymes, these approaches may result in incomplete data. In this paper, we tested commercially available PIs antibodies by light microscopy on fixed cells, tested their specificity using protein-lipid overlay assay and blocking assay, and compared their staining patterns. Additionally, we prepared recombinant PIs-binding domains and tested them on both fixed and live cells by light microscopy. The results provide a useful overview of usability of the tools tested and stress that the selection of adequate tools is critical. Knowing the localization of individual PIs in various functional compartments should enable us to better understand the roles of PIs in the cell nucleus.

Automated system for measuring temperature profiles inside ITS-90 fixed-point cells

NASA Astrophysics Data System (ADS)

Hiti, Miha; Bojkovski, Jovan; Batagelj, Valentin; Drnovsek, Janko

2005-11-01

The defining fixed points of the International Temperature Scale of 1990 (ITS-90) are temperature reference points for temperature calibration. The measured temperature inside the fixed-point cell depends on thermometer immersion, since measurements are made below the surface of the fixed-point material and the additional effect of the hydrostatic pressure has to be taken into account. Also, the heat flux along the thermometer stem can affect the measured temperature. The paper presents a system that enables accurate and reproducible immersion profile measurements for evaluation of measurement sensitivity and adequacy of thermometer immersion. It makes immersion profile measurements possible, where a great number of repetitions and long measurement periods are required, and reduces the workload on the user for performing such measurements. The system is flexible and portable and was developed for application to existing equipment in the laboratory. Results of immersion profile measurements in a triple point of water fixed-point cell are presented.

Comparison of the Argon Triple-Point Temperature in Small Cells of Different Construction

NASA Astrophysics Data System (ADS)

Kołodziej, B.; Kowal, A.; Lipiński, L.; Manuszkiewicz, H.; Szmyrka-Grzebyk, A.

2017-06-01

The argon triple point (T_{90} = 83.8058 \\hbox {K}) is a fixed point of the International Temperature Scale of Preston-Thomas (Metrologia 27:3, 1990). Cells for realization of the fixed point have been manufactured by several European metrology institutes (Pavese in Metrologia 14:93, 1978; Pavese et al. in Temperature, part 1, American Institute of Physics, College Park, 2003; Hermier et al. in Temperature, part 1, American Institute of Physics, College Park, 2003; Pavese and Beciet in Modern gas-based temperature and pressure measurement, Springer, New York, 2013). The Institute of Low Temperature and Structure Research has in its disposal a few argon cells of various constructions used for calibration of capsule-type standard platinum resistance thermometers (CSPRT) that were produced within 40 years. These cells differ in terms of mechanical design and thermal properties, as well as source of gas filling the cell. This paper presents data on differences between temperature values obtained during the realization of the triple point of argon in these cells. For determination of the temperature, a heat-pulse method was applied (Pavese and Beciet in Modern gas-based temperature and pressure measurement, Springer, New York, 2013). The comparisons were performed using three CSPRTs. The temperatures difference was determined in relation to a reference function W(T)=R(T_{90})/R(271.16\\hbox {K}) in order to avoid an impact of CSPRT resistance drift between measurements in the argon cells. Melting curves and uncertainty budgets of the measurements are given in the paper. A construction of measuring apparatus is also presented in this paper.

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines

PubMed Central

Korać, P; Jones, M; Dominis, M; Kušec, R; Mason, D Y; Banham, A H; Ventura, R A

2005-01-01

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease. PMID:16311361

Development of economical improved thick film solar cell contact

NASA Technical Reports Server (NTRS)

Ross, B.

1979-01-01

Metal screened electrodes were investigated with base metal pastes and silver systems being focused upon. Contact resistance measurements were refined. A facility allowing fixing in hydrogen and other atmospheres was acquired. Several experiments were made applying screenable pastes to solar cells. Doping investigations emphasized eutectic alloys reduced to powders. Metal systems were reviewed and base metal experiments were done with nickel and copper using lead and tin as the frit metals. Severe adhesion problems were experienced with hydrogen atmospheres in all metal systems. A two step firing schedule was devised. Aluminum-silicon and aluminum-germanium eutectic doping additions to copper pastes were tried on 2 1/4 in diameter solar cell back contacts, both with good results.

Indirect Determination of the Thermodynamic Temperature of a Gold Fixed-Point Cell

NASA Astrophysics Data System (ADS)

Battuello, M.; Girard, F.; Florio, M.

2010-09-01

Since the value T 90(Au) was fixed on the ITS-90, some determinations of the thermodynamic temperature of the gold point have been performed which form, with other renormalized results of previous measurements by radiation thermometry, the basis for the current best estimates of ( T - T 90)Au = 39.9 mK as elaborated by the CCT-WG4. Such a value, even if consistent with the behavior of T - T 90 differences at lower temperatures, is quite influenced by the low values of T Au as determined with few radiometric measurements. At INRIM, an independent indirect determination of the thermodynamic temperature of gold was performed by means of a radiation thermometry approach. A fixed-point technique was used to realize approximated thermodynamic scales from the Zn point up to the Cu point. A Si-based standard radiation thermometer working at 900 nm and 950 nm was used. The low uncertainty presently associated to the thermodynamic temperature of fixed points and the accuracy of INRIM realizations, allowed scales with an uncertainty lower than 0.03 K in terms of the thermodynamic temperature to be realized. A fixed-point cell filled with gold, 99.999 % in purity, was measured, and its freezing temperature was determined by both interpolation and extrapolation. An average T Au = 1337.395 K was found with a combined standard uncertainty of 23 mK. Such a value is 25 mK higher than the presently available value as derived by the CCT-WG4 value of ( T - T 90)Au = 39.9 mK.

Morphology and force probing of primary murine liver sinusoidal endothelial cells.

PubMed

Zapotoczny, B; Owczarczyk, K; Szafranska, K; Kus, E; Chlopicki, S; Szymonski, M

2017-07-01

Liver sinusoidal endothelial cells (LSECs) represent unique type of endothelial cells featured by their characteristic morphology, ie, lack of a basement membrane and presence of fenestrations-transmembrane pores acting as a dynamic filter between the vascular space and the liver parenchyma. Delicate structure of LSECs membrane combined with a submicron size of fenestrations hinders their visualization in live cells. In this work, we apply atomic force microscopy contact mode to characterize fenestrations in LSECs. We reveal the structure of fenestrations in live LSECs. Moreover, we show that the high-resolution imaging of fenestrations is possible for the glutaraldehyde-fixed LSECs. Finally, thorough information about the morphology of LSECs including great contrast in visualization of sieve plates and fenestrations is provided using Force Modulation mode. We show also the ability to precisely localize the cell nuclei in fixed LSECs. It can be helpful for more precise description of nanomechanical properties of cell nuclei using atomic force microscopy. Presented methodology combining high-quality imaging of fixed cells with an additional nanomechanical information of both live and fixed LSECs provides a unique approach to study LSECs morphology and nanomechanics that could foster understanding of the role of LSECs in maintaining liver homeostasis. Copyright © 2017 John Wiley & Sons, Ltd.

Light absorption cell combining variable path and length pump

DOEpatents

Prather, William S.

1993-01-01

A device for use in making spectrophotometric measurements of fluid samples. In particular, the device is a measurement cell containing a movable and a fixed lens with a sample of the fluid therebetween and through which light shines. The cell is connected to a source of light and a spectrophotometer via optic fibers. Movement of the lens varies the path length and also pumps the fluid into and out of the cell. Unidirectional inlet and exit valves cooperate with the movable lens to assure a one-way flow of fluid through the cell. A linear stepper motor controls the movement of the lens and cycles it from a first position closer to the fixed lens and a second position farther from the fixed lens, preferably at least 10 times per minute for a nearly continuous stream of absorption spectrum data.

Functional Single-Cell Approach to Probing Nitrogen-Fixing Bacteria in Soil Communities by Resonance Raman Spectroscopy with 15N2 Labeling.

PubMed

Cui, Li; Yang, Kai; Li, Hong-Zhe; Zhang, Han; Su, Jian-Qiang; Paraskevaidi, Maria; Martin, Francis L; Ren, Bin; Zhu, Yong-Guan

2018-04-17

Nitrogen (N) fixation is the conversion of inert nitrogen gas (N 2 ) to bioavailable N essential for all forms of life. N 2 -fixing microorganisms (diazotrophs), which play a key role in global N cycling, remain largely obscure because a large majority are uncultured. Direct probing of active diazotrophs in the environment is still a major challenge. Herein, a novel culture-independent single-cell approach combining resonance Raman (RR) spectroscopy with 15 N 2 stable isotope probing (SIP) was developed to discern N 2 -fixing bacteria in a complex soil community. Strong RR signals of cytochrome c (Cyt c, frequently present in diverse N 2 -fixing bacteria), along with a marked 15 N 2 -induced Cyt c band shift, generated a highly distinguishable biomarker for N 2 fixation. 15 N 2 -induced shift was consistent well with 15 N abundance in cell determined by isotope ratio mass spectroscopy. By applying this biomarker and Raman imaging, N 2 -fixing bacteria in both artificial and complex soil communities were discerned and imaged at the single-cell level. The linear band shift of Cyt c versus 15 N 2 percentage allowed quantification of N 2 fixation extent of diverse soil bacteria. This single-cell approach will advance the exploration of hitherto uncultured diazotrophs in diverse ecosystems.

Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B

PubMed Central

Su, Jin; Zhu, Liqing; Sherman, Alexandra; Wang, Xiaomei; Lin, Shina; Kamesh, Aditya; Norikane, Joey H.; Streatfield, Stephen J.; Herzog, Roland W.; Daniell, Henry

2015-01-01

Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (~1mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ~2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP+ regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ~870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft2 per annum yielding 24,000–36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs. PMID:26302233

Method and apparatus for sustaining viability of biological cells on a substrate

DOEpatents

McKnight, Timothy E.; Melechko, Anatoli V.; Simpson, Michael L.

2013-01-01

A method for the transient transformation of a living biological cell having an intact cell membrane defining an intracellular domain, and an apparatus for the transient transformation of biological cells. The method and apparatus include introducing a compartmentalized extracellular component fixedly attached to a cellular penetrant structure to the intracellular domain of the cell, wherein the cell is fixed in a predetermined location and wherein the component is expressed within in the cell while being retained within the compartment and wherein the compartment restricts the mobility and interactions of the component within the cell and prevents transference of the component to the cell.

Method and apparatus for sustaining viability of biological cells on a substrate

DOEpatents

McKnight, Timothy E [Greenback, TN; Melechko, Anatoli V [Oak Ridge, TN; Simpson, Michael L [Knoxville, TN

2011-12-13

A method for the transient transformation of a living biological cell having an intact cell membrane defining an intracellular domain, and an apparatus for the transient transformation of biological cells. The method and apparatus include introducing a compartmentalized extracellular component fixedly attached to a cellular penetrant structure to the intracellular domain of the cell, wherein the cell is fixed in a predetermined location and wherein the component is expressed within in the cell while being retained within the compartment and wherein the compartment restricts the mobility and interactions of the component within the cell and prevents transference of the component to the cell.

Method of fabricating a solar cell array

DOEpatents

Lazzery, Angelo G.; Crouthamel, Marvin S.; Coyle, Peter J.

1982-01-01

A first set of pre-tabbed solar cells are assembled in a predetermined array with at least part of each tab facing upward, each tab being fixed to a bonding pad on one cell and abutting a bonding pad on an adjacent cell. The cells are held in place with a first vacuum support. The array is then inverted onto a second vacuum support which holds the tabs firmly against the cell pads they abut. The cells are exposed to radiation to melt and reflow the solder pads for bonding the tab portions not already fixed to bonding pads to these pads.

Cell morphology and flagellation of nitrogen-fixing spirilla.

PubMed

Hegazi, N A; Vlassak, K

1979-01-01

Twenty isolates of N2-fixing spirilla were isolated from the rhizosphere of maize and sugar cane grown in Egyptian and Belgian soils. Electron microscopy distinguished two morphological groups. The first includes short and thick curved rods with an unipolar flagellum while cells of the second group are much longer with the typical appearance of spiral cells and most probably possess a bipolar tuft of flagella.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

NASA Astrophysics Data System (ADS)

Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

A novel IMSL tunable phase shifter for HMSIW-LWA-fed rectangular patches based on nematic liquid crystal

NASA Astrophysics Data System (ADS)

Fu, JiaHui; Raheem, Odai H.

2017-07-01

A novel IMSL tunable phase shifter for HMSIW-LWA-fed rectangular patches based on liquid crystal technology is proposed. Rectangular patches are used as radiators for the opening sidewall of the waveguide and matched section part for a unit cell. The transition structure is added for enhancing the efficiency of HMSIW-LWA due to converting most input power to the leaky mode. The novel IMSL phase shifter is used for investigating the tunable dielectric characteristics of N-LC by applying an electric field to the LC cell, which is controlled by the orientation angle of the LC molecules. Theoretically, the orientation angle is derived and solved numerically with the accurate method. As a result, the HMSIW-LWA can be tuned up to ± 25° for a fixed frequency by tuning the nematic LC with applied voltage from 0 to 20 V. In addition, the realized gain changed from 6 to 9.4 dB for a fixed tuned frequency, and 46° steerable for rest main beams range of the HMSIW-LWA in both forward and backward directions.

Laser-Induced Melting of Co-C Eutectic Cells as a New Research Tool

NASA Astrophysics Data System (ADS)

van der Ham, E.; Ballico, M.; Jahan, F.

2015-08-01

A new laser-based technique to examine heat transfer and energetics of phase transitions in metal-carbon fixed points and potentially to improve the quality of phase transitions in furnaces with poor uniformity is reported. Being reproducible below 0.1 K, metal-carbon fixed points are increasingly used as reference standards for the calibration of thermocouples and radiation thermometers. At NMIA, the Co-C eutectic point is used for the calibration of thermocouples, with the fixed point traceable to the International Temperature Scale (ITS-90) using radiation thermometry. For thermocouple use, these cells are deep inside a high-uniformity furnace, easily obtaining excellent melting plateaus. However, when used with radiation thermometers, the essential large viewing cone to the crucible restricts the furnace depth and introduces large heat losses from the front furnace zone, affecting the quality of the phase transition. Short laser bursts have been used to illuminate the cavity of a conventional Co-C fixed-point cell during various points in its melting phase transition. The laser is employed to partially melt the metal at the rear of the crucible providing a liquid-solid interface close to the region being observed by the reference pyrometer. As the laser power is known, a quantitative estimate of can be made for the Co-C latent heat of fusion. Using a single laser pulse during a furnace-induced melt, a plateau up to 8 min is observed before the crucible resumes a characteristic conventional melt curve. Although this plateau is satisfyingly flat, well within 100 mK, it is observed that the plateau is laser energy dependent and elevates from the conventional melt "inflection-point" value.

Light absorption cell combining variable path and length pump

DOEpatents

Prather, W.S.

1993-12-07

A device is described for use in making spectrophotometric measurements of fluid samples. In particular, the device is a measurement cell containing a movable and a fixed lens with a sample of the fluid there between and through which light shines. The cell is connected to a source of light and a spectrophotometer via optic fibers. Movement of the lens varies the path length and also pumps the fluid into and out of the cell. Unidirectional inlet and exit valves cooperate with the movable lens to assure a one-way flow of fluid through the cell. A linear stepper motor controls the movement of the lens and cycles it from a first position closer to the fixed lens and a second position farther from the fixed lens, preferably at least 10 times per minute for a nearly continuous stream of absorption spectrum data. 2 figures.

Biocompatibility effects of indirect exposure of base-metal dental casting alloys to a human-derived three-dimensional oral mucosal model.

PubMed

McGinley, Emma Louise; Moran, Gary P; Fleming, Garry J P

2013-11-01

The study employed a three-dimensional (3D) human-derived oral mucosal model to assess the biocompatibility of base-metal dental casting alloys ubiquitous in fixed prosthodontic and orthodontic dentistry. Oral mucosal models were generated using primary human oral keratinocyte and gingival fibroblast cells seeded onto human de-epidermidised dermal scaffolds. Nickel-chromium (Ni-Cr) and cobalt-chromium (Co-Cr) base-metal alloy immersion solutions were exposed to oral mucosal models for increasing time periods (2-72h). Analysis methodologies (histology, viable cell counts, oxidative stress, cytokine expression and toxicity) were performed following exposure. Ni-based alloy immersion solutions elicited significantly decreased cell viability (P<0.0004) with increased oxidative stress (P<0.0053), inflammatory cytokine expression (P<0.0077) and cellular toxicity levels (P<0.0001) compared with the controls. However, the Ni-free Co-Cr-based alloy immersion solutions did not elicit adverse oxidative stress (P>0.4755) or cellular toxicity (P<0.2339) responses compared with controls. Although the multiple analyses highlighted Ni-Cr base-metal alloy immersion solutions elicited significantly detrimental effects to the oral mucosal models, it was possible to distinguish between Ni-Cr alloys using the approach employed. The study employed a 3D human-derived full-thickness differentiated oral mucosal model suitable for biocompatibility assessment of base-metal dental casting alloys through discriminatory experimental parameters. Increasing incidences of Ni hypersensitivity in the general population warrants serious consideration from dental practitioners and patients alike where fixed prosthodontic/orthodontic dental treatments are the treatment modality involved. The novel and analytical oral mucosal model has the potential to significantly contribute to the advancement of reproducible dental medical device and dental material appraisals. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fourier transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium Azospirillum brasilense

NASA Astrophysics Data System (ADS)

Kamnev, A. A.; Ristić, M.; Antonyuk, L. P.; Chernyshev, A. V.; Ignatov, V. V.

1997-06-01

The data of Fourier transform infrared (FTIR) spectroscopic measurements performed on intact cells of the soil nitrogen-fixing bacterium Azospirillum brasilense grown in a standard medium and under the conditions of an increased metal uptake are compared and discussed. The structural FTIR information obtained is considered together with atomic absorption spectrometry (AAS) data on the content of metal cations in the bacterial cells. Some methodological aspects concerning preparation of bacterial cell samples for FTIR measurements are also discussed.

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction.

PubMed

Tubby, Carolyn; Negm, Ola H; Harrison, Timothy; Tighe, Patrick J; Todd, Ian; Fairclough, Lucy C

2017-06-01

The three main types of killer cells - CD8 + T cells, NK cells and NKT cells - have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. Peripheral CD8 + T cells (CD8 + CD3 + CD56 - ), NK cells (CD56 + CD3 - ) and NKT-like cells (CD56 + CD3 + ) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.

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.

Modeling the Chagas’ disease after stem cell transplantation

NASA Astrophysics Data System (ADS)

Galvão, Viviane; Miranda, José Garcia Vivas

2009-04-01

A recent model for Chagas’ disease after stem cell transplantation is extended for a three-dimensional multi-agent-based model. The computational model includes six different types of autonomous agents: inflammatory cell, fibrosis, cardiomyocyte, proinflammatory cytokine tumor necrosis factor- α, Trypanosoma cruzi, and bone marrow stem cell. Only fibrosis is fixed and the other types of agents can move randomly through the empty spaces using the three-dimensional Moore neighborhood. Bone marrow stem cells can promote apoptosis in inflammatory cells, fibrosis regression and can differentiate in cardiomyocyte. T. cruzi can increase the number of inflammatory cells. Inflammatory cells and tumor necrosis factor- α can increase the quantity of fibrosis. Our results were compared with experimental data giving a fairly fit and they suggest that the inflammatory cells are important for the development of fibrosis.

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

PubMed

Ishikawa, Tomomi; Kitano, Hisataka; Mamiya, Atsushi; Kokubun, Shinichiro; Hidai, Chiaki

2016-07-01

Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner. © 2016 The Author(s).

Influence of the Cavity Length on the Behavior of Hybrid Fixed-Point Cells Constructed at INRIM

NASA Astrophysics Data System (ADS)

Battuello, M.; Girard, F.; Florio, M.

2015-03-01

Hybrid cells with double carbon/carbon sheets are used at the Istituto Nazionale di Ricerca Metrologica (INRIM) for the realization of both pure metal fixed points and high-temperature metal-carbon eutectic points. Cells for the Cu and Co-C fixed points have been prepared to be used in the high-temperature fixed-point project of the Comité Consultatif de Thermométrie. The results of the evaluation processes were not completely satisfactory for the INRIM cells because of their low transition temperatures with respect to the best cells, and of a rather large melting range for the Co-C cell. A new design of the cells was devised, and considerable improvements were achieved with respect to the transition temperature, and the plateau shape and duration. As for the Cu point, the duration of the freezing plateaux increased by more than 50 % and the freezing temperature increased by 18 mK. As for the Co-C point, the melting temperature, expressed in terms of the point of inflection of the melting curve, increased by about 70 mK. The melting range of the plateaux, expressed as a difference was reduced from about 180 mK to about 130 mK, with melting times increased by about 50 %, as a consequence of an improvement of flatness and run-off of the plateaux.

Polymerase chain reaction-based detection of B-cell monoclonality in cytologic specimens.

PubMed

Chen, Y T; Mercer, G O; Chen, Y

1993-11-01

Thirty-seven cytologic cell blocks were evaluated for B-cell monoclonality by polymerase chain reaction (PCR), 16 of them cytologically positive for lymphoma, and 21 suspicious for lymphoma but morphologically nondiagnostic. Of 37 specimens, 13 (35%) showed B-cell monoclonality, including six of 16 cytologically positive samples and seven of 21 cytologically suspicious ones. Of these 13 positive samples, seven were positive using crude lysates as substrates, and six additional positive samples were identified only when DNAs were purified and concentrated. Analysis of the DNAs further revealed poor polymerase chain reaction amplifiability and low DNA yield in many samples, indicating that cell block materials are suboptimal for this assay. We concluded that B-cell monoclonality can be detected in ethanol-fixed cytologic samples, and usage of unembedded material will likely improve the sensitivity. In specimens cytologically suspicious for lymphoma, polymerase chain reaction-based identification of monoclonal B-cell population supports the diagnosis of B-cell lymphoma and is a potentially useful test in solving this diagnostic dilemma.

Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B.

PubMed

Su, Jin; Zhu, Liqing; Sherman, Alexandra; Wang, Xiaomei; Lin, Shina; Kamesh, Aditya; Norikane, Joey H; Streatfield, Stephen J; Herzog, Roland W; Daniell, Henry

2015-11-01

Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The upper surface of an Escherichia coli swarm is stationary.

PubMed

Zhang, Rongjing; Turner, Linda; Berg, Howard C

2010-01-05

When grown in a rich medium on agar, many bacteria elongate, produce more flagella, and swim in a thin film of fluid over the agar surface in swirling packs. Cells that spread in this way are said to swarm. The agar is a solid gel, with pores smaller than the bacteria, so the swarm/agar interface is fixed. Here we show, in experiments with Escherichia coli, that the swarm/air interface also is fixed. We deposited MgO smoke particles on the top surface of an E. coli swarm near its advancing edge, where cells move in a single layer, and then followed the motion of the particles by dark-field microscopy and the motion of the underlying cells by phase-contrast microscopy. Remarkably, the smoke particles remained fixed (diffusing only a few micrometers) while the swarming cells streamed past underneath. The diffusion coefficients of the smoke particles were smaller over the virgin agar ahead of the swarm than over the swarm itself. Changes between these two modes of behavior were evident within 10-20 microm of the swarm edge, indicating an increase in depth of the fluid in advance of the swarm. The only plausible way that the swarm/air interface can be fixed is that it is covered by a surfactant monolayer pinned at its edges. When a swarm is exposed to air, such a monolayer can markedly reduce water loss. When cells invade tissue, the ability to move rapidly between closely opposed fixed surfaces is a useful trait.

Large-scale Clinical-grade Retroviral Vector Production in a Fixed-Bed Bioreactor

PubMed Central

Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel

2015-01-01

The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

Implementation of stimulated Raman scattering microscopy for single cell analysis

NASA Astrophysics Data System (ADS)

D'Arco, Annalisa; Ferrara, Maria Antonietta; Indolfi, Maurizio; Tufano, Vitaliano; Sirleto, Luigi

2017-05-01

In this work, we present successfully realization of a nonlinear microscope, not purchasable in commerce, based on stimulated Raman scattering. It is obtained by the integration of a femtosecond SRS spectroscopic setup with an inverted research microscope equipped with a scanning unit. Taking account of strength of vibrational contrast of SRS, it provides label-free imaging of single cell analysis. Validation tests on images of polystyrene beads are reported to demonstrate the feasibility of the approach. In order to test the microscope on biological structures, we report and discuss the label-free images of lipid droplets inside fixed adipocyte cells.

Dynamic morphology applied to human and animal leukemia cells.

PubMed

Haemmerli, G; Felix, H; Sträuli, P

1979-08-01

Dynamic morphology, which describes the shape and surface architecture of fixed cells in terms related to their behavior in the living state, is based on the concurrent use of two methods: scanning electron microscopy and microcinematography. This combination has both advantages and disadvantages. In this study on leukemic cells, we were able to draw the following conclusions about the usefulness of dynamic morphology. It confirms that white blood cells do not flatten on a glass substrate; they stay spherical and are either round or polarized. Round cells of similar size, whatever their origin, cannot be classified by dynamic morphology. Polarized cells can be classified as blasts, promyelocytes, myelocytes, granulocytes and lymphocytes, although polarized blast cells of different origins cannot be differentiated. Dynamic morphology cannot classify the same cell type as benign or malignant.

Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.

PubMed

Jolly, Clare; Mitar, Ivonne; Sattentau, Quentin J

2007-06-01

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

Hydrodynamic interaction of two particles in confined linear shear flow at finite Reynolds number

NASA Astrophysics Data System (ADS)

Yan, Yiguang; Morris, Jeffrey F.; Koplik, Joel

2007-11-01

We discuss the hydrodynamic interactions of two solid bodies placed in linear shear flow between parallel plane walls in a periodic geometry at finite Reynolds number. The computations are based on the lattice Boltzmann method for particulate flow, validated here by comparison to previous results for a single particle. Most of our results pertain to cylinders in two dimensions but some examples are given for spheres in three dimensions. Either one mobile and one fixed particle or else two mobile particles are studied. The motion of a mobile particle is qualitatively similar in both cases at early times, exhibiting either trajectory reversal or bypass, depending upon the initial vector separation of the pair. At longer times, if a mobile particle does not approach a periodic image of the second, its trajectory tends to a stable limit point on the symmetry axis. The effect of interactions with periodic images is to produce nonconstant asymptotic long-time trajectories. For one free particle interacting with a fixed second particle within the unit cell, the free particle may either move to a fixed point or take up a limit cycle. Pairs of mobile particles starting from symmetric initial conditions are shown to asymptotically reach either fixed points, or mirror image limit cycles within the unit cell, or to bypass one another (and periodic images) indefinitely on a streamwise periodic trajectory. The limit cycle possibility requires finite Reynolds number and arises as a consequence of streamwise periodicity when the system length is sufficiently short.

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

PubMed Central

Kumar, Sunil; Alibhai, Dominic; Margineanu, Anca; Laine, Romain; Kennedy, Gordon; McGinty, James; Warren, Sean; Kelly, Douglas; Alexandrov, Yuriy; Munro, Ian; Talbot, Clifford; Stuckey, Daniel W; Kimberly, Christopher; Viellerobe, Bertrand; Lacombe, Francois; Lam, Eric W-F; Taylor, Harriet; Dallman, Margaret J; Stamp, Gordon; Murray, Edward J; Stuhmeier, Frank; Sardini, Alessandro; Katan, Matilda; Elson, Daniel S; Neil, Mark A A; Dunsby, Chris; French, Paul M W

2011-01-01

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated. PMID:21337485

3D time-lapse analysis of Rab11/FIP5 complex: spatiotemporal dynamics during apical lumen formation.

PubMed

Mangan, Anthony; Prekeris, Rytis

2015-01-01

Fluorescent imaging of fixed cells grown in two-dimensional (2D) cultures is one of the most widely used techniques for observing protein localization and distribution within cells. Although this technique can also be applied to polarized epithelial cells that form three-dimensional (3D) cysts when grown in a Matrigel matrix suspension, there are still significant limitations in imaging cells fixed at a particular point in time. Here, we describe the use of 3D time-lapse imaging of live cells to observe the dynamics of apical membrane initiation site (AMIS) formation and lumen expansion in polarized epithelial cells.

Electrophoretic cell separation by means of immunomicrospheres

NASA Technical Reports Server (NTRS)

Rembaum, A.; Smolka, A. J. K.

1980-01-01

The electrophoretic mobility of fixed human red blood cells immunologically labeled with polymeric (4-vinyl)pyridine or polyglutaraldehyde microspheres was altered to a considerable extent. This observation was utilized in the preparative scale electrophoretic separation of human and turkey fixed red blood cells, whose mobilities under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. It is suggested that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immuno-specific labeling of target populations using microspheres.

Trace metal ions release from fixed orthodontic appliances and DNA damage in oral mucosa cells by in vivo studies: A literature review.

PubMed

Downarowicz, Patrycja; Mikulewicz, Marcin

2017-10-01

An overview of professional literature referring to the release of metal ions from fixed orthodontic appliances and their influence on oral mucosa in conditions of in vivo are presented, along with a detailed analysis of the exposure of the cells of cheek mucosa epithelium to metal ions. Electronic databases (PubMed, Elsevier, Ebsco) were searched with no language restrictions. The relevant orthodontic journals and reference lists were checked for all eligible studies. A total of 38 scientific articles were retrieved in the initial search. However, only 7 articles met the inclusion criteria. Statistically significant differences in the levels of the amount of nickel ions, cobalt ions and chromium ions were observed in cells of cheek mucosa. The most biocompatible material used in the production of fixed orthodontic appliances is titanium, and the least biocompatible material is steel, which releases the largest amount of nickel and chromium. Metal ions are released from fixed orthodontic appliances only in the first phase of treatment. It is recommended to conduct further, long-term research on a larger number of patients to define the influence of using fixed orthodontic appliances and biological effect they might have on tissues.

Effects of Lugol's iodine solution and formalin on cell volume of three bloom-forming dinoflagellates

NASA Astrophysics Data System (ADS)

Yang, Yang; Sun, Xiaoxia; Zhao, Yongfang

2017-07-01

Fixatives are traditionally used in marine ecosystem research. The bias introduced by fixatives on the dimensions of plankton cells may lead to an overestimation or underestimation of the carbon biomass. To determine the impact of traditional fixatives on dinoflagellates during short- and long-term fixation, we analyzed the degree of change in three bloom-forming dinoflagellates ( Prorocentrum micans, Scrippsiella trochoidea and Noctiluca scintillans) brought about by Lugol's iodine solution (hereafter Lugol's) and formalin. The fixation effects were species-specific. P. micans cell volume showed no significant change following long-term preservation, and S. trochoidea swelled by approximately 8.06% in Lugol's and by 20.97% in formalin as a percentage of the live cell volume, respectively. N. scintillans shrank significantly in both fixatives. The volume change due to formalin in N. scintillans was not concentration-dependent, whereas the volume shrinkage of N. scintillans cells fixed with Lugol's at a concentration of 2% was nearly six-fold that in cells fixed with Lugol's at a concentration of 0.6%-0.8%. To better estimate the volume of N. scintillans fixed in formalin at a concentration of 5%, we suggest that the conversion relationship was as follows: volume of live cell=volume of intact fixed cell/0.61. Apart from size change, damage induced by fixatives on N. scintillans was obvious. Lugol's is not a suitable fixative for N. scintillans due to high frequency of broken cells. Accurate carbon biomass estimate of N. scintillans should be performed on live samples. These findings help to improve the estimate of phytoplankton cell volume and carbon biomass in marine ecosystem.

Nanomedicine: nanoparticles, molecular biosensors, and targeted gene/drug delivery for combined single-cell diagnostics and therapeutics

NASA Astrophysics Data System (ADS)

Prow, Tarl W.; Salazar, Jose H.; Rose, William A.; Smith, Jacob N.; Reece, Lisa; Fontenot, Andrea A.; Wang, Nan A.; Lloyd, R. Stephen; Leary, James F.

2004-07-01

Next generation nanomedicine technologies are being developed to provide for continuous and linked molecular diagnostics and therapeutics. Research is being performed to develop "sentinel nanoparticles" which will seek out diseased (e.g. cancerous) cells, enter those living cells, and either perform repairs or induce those cells to die through apoptosis. These nanoparticles are envisioned as multifunctional "smart drug delivery systems". The nanosystems are being developed as multilayered nanoparticles (nanocrystals, nanocapsules) containing cell targeting molecules, intracellular re-targeting molecules, molecular biosensor molecules, and drugs/enzymes/gene therapy. These "nanomedicine systems" are being constructed to be autonomous, much like present-day vaccines, but will have sophisticated targeting, sensing, and feedback control systems-much more sophisticated than conventional antibody-based therapies. The fundamental concept of nanomedicine is to not to just kill all aberrant cells by surgery, radiation therapy, or chemotherapy. Rather it is to fix cells, when appropriate, one cell-at-a-time, to preserve and re-build organ systems. When cells should not be fixed, such as in cases where an improperly repaired cell might give rise to cancer cells, the nanomedical therapy would be to induce apoptosis in those cells to eliminate them without the damagin bystander effects of the inflammatory immune response system reacting to necrotic cells or those which have died from trauma or injury. The ultimate aim of nanomedicine is to combine diagnostics and therapeutics into "real-time medicine", using where possible in-vivo cytometry techniques for diagnostics and therapeutics. A number of individual components of these multi-component nanoparticles are already working in in-vitro and ex-vivo cell and tissue systems. Work has begun on construction of integrated nanomedical systems.

Modeling the role of quorum sensing in interspecies competition in biofilms

NASA Astrophysics Data System (ADS)

Narla, Avaneesh V.; Wingreen, Ned S.; Borenstein, David B.

Bacteria grow on surfaces in complex immobile communities known as biofilms, composed of cells embedded in an extracellular matrix. Within biofilms, bacteria often communicate, cooperate, and compete within their own species and with other species using Quorum Sensing (QS). QS refers to the process by which bacteria produce, secrete, and subsequently detect small molecules called autoinducers as a way to assess the local population density of their species, or of other species. QS is known to regulate the production of extracellular matrix. We investigated the possible benefit of QS in regulating matrix production to best gain access to a nutrient that diffuses from a source positioned away from the surface on which the biofilm grows. We employed Agent-Based Modeling (ABM), a form of simulation that allows cells to modify their behavior based on local inputs, e.g. nutrient and QS concentrations. We first determined the optimal fixed strategies (that do not use QS) for pairwise competitions, and then demonstrated that simple QS-based strategies can be superior to any fixed strategy. In nature, species can compete by sensing and/or interfering with each other's QS signals, and we explore approaches for targeting specific species via QS-interference. A.V.N. and N.S.W. contributed equally to this project.

Realization of the Temperature Scale in the Range from 234.3 K (Hg Triple Point) to 1084.62°C (Cu Freezing Point) in Croatia

NASA Astrophysics Data System (ADS)

Zvizdic, Davor; Veliki, Tomislav; Grgec Bermanec, Lovorka

2008-06-01

This article describes the realization of the International Temperature Scale in the range from 234.3 K (mercury triple point) to 1084.62°C (copper freezing point) at the Laboratory for Process Measurement (LPM), Faculty of Mechanical Engineering and Naval Architecture (FSB), University of Zagreb. The system for the realization of the ITS-90 consists of the sealed fixed-point cells (mercury triple point, water triple point and gallium melting point) and the apparatus designed for the optimal realization of open fixed-point cells which include the gallium melting point, tin freezing point, zinc freezing point, aluminum freezing point, and copper freezing point. The maintenance of the open fixed-point cells is described, including the system for filling the cells with pure argon and for maintaining the pressure during the realization.

On the application of the PFEM to droplet dynamics modeling in fuel cells

NASA Astrophysics Data System (ADS)

Ryzhakov, Pavel B.; Jarauta, Alex; Secanell, Marc; Pons-Prats, Jordi

2017-07-01

The Particle Finite Element Method (PFEM) is used to develop a model to study two-phase flow in fuel cell gas channels. First, the PFEM is used to develop the model of free and sessile droplets. The droplet model is then coupled to an Eulerian, fixed-grid, model for the airflow. The resulting coupled PFEM-Eulerian algorithm is used to study droplet oscillations in an air flow and droplet growth in a low-temperature fuel cell gas channel. Numerical results show good agreement with predicted frequencies of oscillation, contact angle, and deformation of injected droplets in gas channels. The PFEM-based approach provides a novel strategy to study droplet dynamics in fuel cells.

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

32 CFR 37.300 - What is the difference between an expenditure-based and fixed-support TIA?

Code of Federal Regulations, 2010 CFR

2010-07-01

... SECRETARY OF DEFENSE DoD GRANT AND AGREEMENT REGULATIONS TECHNOLOGY INVESTMENT AGREEMENTS Expenditure-Based and Fixed-Support Technology Investment Agreements § 37.300 What is the difference between an expenditure-based and fixed-support TIA? The fundamental difference between an expenditure-based and fixed...

Androgen Deprivation Enhances PLZF-Repressed Cistrome that Promotes the Castration-Resistant Phenotype

DTIC Science & Technology

2014-10-01

Briefly, 10 million cells were used per IP. Cells were fixed with 1% formaldehyde solution. DNA was sonicated and subjected to immunoprecipitation with... formaldehyde solution. Total chromatin and RNAs were sonicated and subjected to immunoprecipitation with the same AR and Med1 antibodies used in ChIP...were fixed with 1% formaldehyde . Cell pellets were lysed and resuspended in restriction buffer for BstY1 and 0.1% SDS for 10 min at 65 °C. Triton X

Identification of 50- and 23-/25-kDa HeLa cell membrane glycoproteins involved in poliovirus infection: occurrence of poliovirus specific binding sites on susceptible and nonsusceptible cells.

PubMed

Barnert, R H; Zeichhardt, H; Habermehl, K O

1992-02-01

Glycoproteins in the range 50 and 23/25 kDa were identified as poliovirus specific binding sites on HeLa cells with the monoclonal antibody mAb 122. mAb 122 is characterized by its partial inhibiting effect on poliovirus reproduction and adsorption when prebound to HeLa cells. The binding sites are endocytosed in native cells and specific for poliovirus as mAb 122 did not interfere with the adsorption of human rhinovirus type 14 (HRV 14). The poliovirus binding sites are present also on nonprimate so called nonsusceptible cells, e.g., mouse L-cells, as could be shown with sensitive ELISA based binding assays and performance of binding studies with fixed cells at 37 degrees.

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers.

PubMed

Cocking, Edward C; Stone, Philip J; Davey, Michael R

2005-12-01

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers.

PubMed

Cocking, Edward C; Stone, Philip J; Davey, Michael R

2005-09-01

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium,Gluconacetobacter diazotrophicus that naturally occurs in sugarcane.G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization byG. diazotrophicus, with minimal or zero inputs.

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Evidence is presented to show that guinea pig complement fixed on sensitized sheep red cells acts as a specific agglutinogen. Agglutinating antibodies that react with cell-fixed complement can be produced by immunizing rabbits with a complex of stromata-amboceptor-complement or with guinea pig serum globulin. These agglutinins can be removed by precipitation with guinea pig serum. They are, therefore, distinct from immunoconglutinins. PMID:14409702

Identification and genetic analysis of cancer cells with PCR-activated cell sorting

PubMed Central

Eastburn, Dennis J.; Sciambi, Adam; Abate, Adam R.

2014-01-01

Cell sorting is a central tool in life science research for analyzing cellular heterogeneity or enriching rare cells out of large populations. Although methods like FACS and FISH-FC can characterize and isolate cells from heterogeneous populations, they are limited by their reliance on antibodies, or the requirement to chemically fix cells. We introduce a new cell sorting technology that robustly sorts based on sequence-specific analysis of cellular nucleic acids. Our approach, PCR-activated cell sorting (PACS), uses TaqMan PCR to detect nucleic acids within single cells and trigger their sorting. With this method, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132 000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Additionally, we demonstrate that PACS can be used to sort and enrich cells via TaqMan PCR reactions targeting single-copy genomic DNA. PACS provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches. PMID:25030902

A Comprehensively Curated Genome-Scale Two-Cell Model for the Heterocystous Cyanobacterium Anabaena sp. PCC 71201[CC-BY

PubMed Central

Steuer, Ralf

2017-01-01

Anabaena sp. PCC 7120 is a nitrogen-fixing filamentous cyanobacterium. Under nitrogen-limiting conditions, a fraction of the vegetative cells in each filament terminally differentiate to nongrowing heterocysts. Heterocysts are metabolically and structurally specialized to enable O2-sensitive nitrogen fixation. The functionality of the filament, as an association of vegetative cells and heterocysts, is postulated to depend on metabolic exchange of electrons, carbon, and fixed nitrogen. In this study, we compile and evaluate a comprehensive curated stoichiometric model of this two-cell system, with the objective function based on the growth of the filament under diazotrophic conditions. The predicted growth rate under nitrogen-replete and -deplete conditions, as well as the effect of external carbon and nitrogen sources, was thereafter verified. Furthermore, the model was utilized to comprehensively evaluate the optimality of putative metabolic exchange reactions between heterocysts and vegetative cells. The model suggested that optimal growth requires at least four exchange metabolites. Several combinations of exchange metabolites resulted in predicted growth rates that are higher than growth rates achieved by only considering exchange of metabolites previously suggested in the literature. The curated model of the metabolic network of Anabaena sp. PCC 7120 enhances our ability to understand the metabolic organization of multicellular cyanobacteria and provides a platform for further study and engineering of their metabolism. PMID:27899536

PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

PubMed Central

Mollenhauer, Hilton H.; Zebrun, William

1960-01-01

Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

In-silico analysis on biofabricating vascular networks using kinetic Monte Carlo simulations.

PubMed

Sun, Yi; Yang, Xiaofeng; Wang, Qi

2014-03-01

We present a computational modeling approach to study the fusion of multicellular aggregate systems in a novel scaffold-less biofabrication process, known as 'bioprinting'. In this novel technology, live multicellular aggregates are used as fundamental building blocks to make tissues or organs (collectively known as the bio-constructs,) via the layer-by-layer deposition technique or other methods; the printed bio-constructs embedded in maturogens, consisting of nutrient-rich bio-compatible hydrogels, are then placed in bioreactors to undergo the cellular aggregate fusion process to form the desired functional bio-structures. Our approach reported here is an agent-based modeling method, which uses the kinetic Monte Carlo (KMC) algorithm to evolve the cellular system on a lattice. In this method, the cells and the hydrogel media, in which cells are embedded, are coarse-grained to material's points on a three-dimensional (3D) lattice, where the cell-cell and cell-medium interactions are quantified by adhesion and cohesion energies. In a multicellular aggregate system with a fixed number of cells and fixed amount of hydrogel media, where the effect of cell differentiation, proliferation and death are tactically neglected, the interaction energy is primarily dictated by the interfacial energy between cell and cell as well as between cell and medium particles on the lattice, respectively, based on the differential adhesion hypothesis. By using the transition state theory to track the time evolution of the multicellular system while minimizing the interfacial energy, KMC is shown to be an efficient time-dependent simulation tool to study the evolution of the multicellular aggregate system. In this study, numerical experiments are presented to simulate fusion and cell sorting during the biofabrication process of vascular networks, in which the bio-constructs are fabricated via engineering designs. The results predict the feasibility of fabricating the vascular structures via the bioprinting technology and demonstrate the morphological development process during cellular aggregate fusion in various engineering designed structures. The study also reveals that cell sorting will perhaps not significantly impact the final fabricated products, should the maturation process be well-controlled in bioprinting.

Reconstruction of the right ventricular outflow tract with a bovine jugular vein graft fixed with a naturally occurring crosslinking agent (genipin) in a canine model.

PubMed

Chang, Y; Tsai, C C; Liang, H C; Sung, H W

2001-12-01

This study was designed to evaluate a newly developed biologic valved conduit fixed with genipin used to reconstruct the right ventricular outflow tract in a canine model. Fresh bovine jugular veins with a retained native valve procured from a slaughterhouse were used as raw materials to fabricate the valved conduits. A naturally occurring crosslinking agent, genipin, was used to fix the procured jugular veins. The glutaraldehyde-fixed counterpart was used as a control. A canine model was used in the study. Echocardiography revealed that the motion of the valvular leaflets in both the glutaraldehyde- and genipin-fixed conduits was satisfactory. The transvalvular pressure gradients of both studied groups were minimal. No endothelium-like cells were observed on the luminal surface of the conduit and the valvular leaflet for the glutaraldehyde-fixed group throughout the entire course of the study. In contrast, endothelium-like cells were observed on the entire surface of the genipin-fixed valved conduit retrieved at 6 months postoperatively in all the cases studied. There was no evidence of luminal fibrous peel in any the valved conduits studied. Degradation of valvular leaflet in one of the glutaraldehyde-fixed conduits was observed. In this particular case, thrombus formation was also observed on the surface of the valvular leaflet. On the other hand, no apparent degradation or thrombus formation was observed on the surfaces of the genipin-fixed valvular leaflet and conduit. A significantly more severe inflammatory reaction was observed for the glutaraldehyde-fixed conduit than for its genipin-fixed counterpart throughout the entire course of the study. The calcium contents of the samples before implantation and those retrieved at distinct implantation duration were minimal for both the glutaraldehyde- and genipin-fixed tissues. Although further studies are necessary, the genipin-fixed valved conduit appears to have great potential in helping mitigate the complications observed in the commercially available conduits.

The Combined Use of a Gas-Controlled Heat Pipe and a Copper Point to Improve the Calibration of Thermocouples up to 1100 ˚C

NASA Astrophysics Data System (ADS)

Astrua, M.; Iacomini, L.; Battuello, M.

2008-10-01

The calibration of platinum-based thermocouples from 420 °C to 1,100 ˚C is currently carried out at INRIM making use of two different apparatus: for temperatures below 930 ˚C, a potassium gas-controlled heat pipe (GCHP) is used, whereas a metal-block furnace is adopted for higher temperatures. The standard uncertainty of the reference temperature obtained in the lower temperature range is almost one order of magnitude better than in the higher temperature range. A sealed copper cell was investigated to see if it could be used to calibrate thermocouples above 930 ˚C with a lower uncertainty than our current procedures allowed. The cell was characterized with Type S and Pt/Pd thermocouples and with an HTPRT. The freezing plateaux were flat within 0.01 ˚C and lasted up to 1 h with a repeatability of 0.02 ˚C. The temperature of the cell was determined with a standard uncertainty of 0.04 ˚C. Hence, the copper cell was found to be superior to the comparator furnace for the calibration of platinum-based thermocouples because of the significant decrease in the uncertainty that it provides. An analysis was also carried out on the calibration of Pt/Pd thermocouples, and it was found that the combined use of the potassium GCHP and the Cu fixed-point cell is adequate to exploit the potential of these sensors in the range from 420 °C to 1,084 °C. A comparison with a fixed-point calibration was also made which gave rise to agreement within 0.07 ˚C between the two approaches.

Ballistic delivery of dyes for structural and functional studies of the nervous system

PubMed Central

Gan, Wen-Biao; Grutzendler, Jaime; Wong, Rachel O.; Lichtman, Jeff W.

2010-01-01

This chapter describes a detail protocol for rapid labeling of cells in a variety of preparations by means of particle-mediated ballistic (gene gun) delivery of fluorescent dyes. This method has been used for rapid labeling of cells with either lipid or water-soluble dyes in a variety of preparations. In particular, carbocyanine lipophilic dyes such as DiI have been used to obtain Golgi-like labeling of neurons and glia in fixed and live cell cultures, brain slices, as well as fixed post-mortem human brain. Water-soluble calcium indicators such as calcium green-1 dextran have been used to image calcium dynamics in living brain slices and retinal explants. This ballistic labeling technique is thus useful for studying the structure and function of neurons and glia in both living and fixed specimens. PMID:20147144

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

PubMed

Dejmek, Annika; Zendehrokh, Nooreldin; Tomaszewska, Malgorzata; Edsjö, Anders

2013-07-01

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society. © 2013 American Cancer Society.

Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.

PubMed

Sandstedt, Mikael; Jonsson, Marianne; Asp, Julia; Dellgren, Göran; Lindahl, Anders; Jeppsson, Anders; Sandstedt, Joakim

2015-12-01

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.

A DIRECT LIGHT EFFECT ON MAINTAINING PHOTOSYNTHETIC ACTIVITY OF NITELLA CHLOROPLASTS

PubMed Central

Craig, I. W.; Gibor, A.

1970-01-01

The chloroplasts of internodal cells of Nitella are fixed to a stationary layer of cytoplasm whereas the nuclei and most of the cytoplasm stream along the longitudinal axis. Isolated internodal cells were maintained for several days with half the cell kept in the dark, the other half kept under continuous light. Photosynthetic activity of the cells was checked by placing the cell evenly illuminated in a 14CO2 atmosphere. Chloroplasts of the previously dark half of the cell were found to fix only half as much CO2 as the chloroplasts which were continuously illuminated. These results are discussed in relation to the possible direct effect of light on biosynthetic reactions of mature chloroplasts. PMID:5411077

A novel histological technique for distinguishing between epithelial cells in forensic casework.

PubMed

French, Claire E V; Jensen, Cynthia G; Vintiner, Susan K; Elliot, Douglas A; McGlashan, Susan R

2008-06-10

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

Experimental and theoretical analysis for improved microscope design of optical projection tomographic microscopy.

PubMed

Coe, Ryan L; Seibel, Eric J

2013-09-01

We present theoretical and experimental results of axial displacement of objects relative to a fixed condenser focal plane (FP) in optical projection tomographic microscopy (OPTM). OPTM produces three-dimensional, reconstructed images of single cells from two-dimensional projections. The cell rotates in a microcapillary to acquire projections from different perspectives where the objective FP is scanned through the cell while the condenser FP remains fixed at the center of the microcapillary. This work uses a combination of experimental and theoretical methods to improve the OPTM instrument design.

Electrophoretic purification of cells in space - Evaluation of results from STS-3

NASA Technical Reports Server (NTRS)

Sarnoff, B. E.; Kunze, M. E.; Todd, P.

1983-01-01

The procedure and results of Electrophoresis Equipment Verification Test, designed to examine electrophoretic behavior of animal cells is suspension more concentrated than possible on earth and flown on the Shuttle flight STS-3, were discussed. Ground-based laboratory values of electrophoretic mobilities of a mixture of human and rabbit aldehyde-fixed red blood cells (RBC) were compared with those recorded at 11 minute intervals on the Shuttle STS-3. RBC migration and separation observed through photographic records were not as expected. However, cell mobilities and migrating band profiles were consistent with the results of laboratory simulation experiments. It was concluded that zero G electrophoresis of very high concentrations (1 x 10 to the 9th) is possible and similar to electrophoresis of normal cell concentrations on earth.

Role of Macrophage-Induced Inflammation in Mesothelioma

DTIC Science & Technology

2010-07-01

in human mesothelioma tumors and correlate immune cell infiltration with histopathologic subtype (months 1-6). Using tumor tissue microarrays of... histopathologic subtype (months 1-6). • Acquired 71 fixed and paraffin-embedded mesothelioma tumor samples • Prepared mesothelioma tumor tissue...Biol., 2008. 84: p. 1-8. 5. Dave, S.S., et al., Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating

ACVP-03: Novel CD4+ T Cell Specific Immunohistochemistry Detection and Analysis Utilizing Masking of Not-T Cell CD4 in Fixed Tissues from Virally Infected and Uninfected Specimens | Frederick National Laboratory for Cancer Research

Cancer.gov

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. CD4 (DAB) and CD68/CD163 (FastRed) double immunohistochemistry will be performed, in whic

Imaging of cellular spread on a three-dimensional scaffold by means of a novel cell-labeling technique for high-resolution computed tomography.

PubMed

Thimm, Benjamin W; Hofmann, Sandra; Schneider, Philipp; Carretta, Roberto; Müller, Ralph

2012-03-01

Computed tomography (CT) represents a truly three-dimensional (3D) imaging technique that can provide high-resolution images on the cellular level. Thus, one approach to detect single cells is X-ray absorption-based CT, where cells are labeled with a dense, opaque material providing the required contrast for CT imaging. Within the present work, a novel cell-labeling method has been developed showing the feasibility of labeling fixed cells with iron oxide (FeO) particles for subsequent CT imaging and quantitative morphometry. A biotin-streptavidin detection system was exploited to bind FeO particles to its target endothelial cells. The binding of the particles was predominantly close to the cell centers on 2D surfaces as shown by light microscopy, scanning electron microscopy, and CT. When cells were cultured on porous, 3D polyurethane surfaces, significantly more FeO particles were detected compared with surfaces without cells and FeO particle labeling using CT. Here, we report on the implementation and evaluation of a novel cell detection method based on high-resolution CT. This system has potential in cell tracking for 3D in vitro imaging in the future.

Lithium-ion Open Circuit Voltage (OCV) curve modelling and its ageing adjustment

NASA Astrophysics Data System (ADS)

Lavigne, L.; Sabatier, J.; Francisco, J. Mbala; Guillemard, F.; Noury, A.

2016-08-01

This paper is a contribution to lithium-ion batteries modelling taking into account aging effects. It first analyses the impact of aging on electrode stoichiometry and then on lithium-ion cell Open Circuit Voltage (OCV) curve. Through some hypotheses and an appropriate definition of the cell state of charge, it shows that each electrode equilibrium potential, but also the whole cell equilibrium potential can be modelled by a polynomial that requires only one adjustment parameter during aging. An adjustment algorithm, based on the idea that for two fixed OCVs, the state of charge between these two equilibrium states is unique for a given aging level, is then proposed. Its efficiency is evaluated on a battery pack constituted of four cells.

Nonequilibrium Population Dynamics of Phenotype Conversion of Cancer Cells

PubMed Central

Zhou, Joseph Xu; Pisco, Angela Oliveira; Qian, Hong; Huang, Sui

2014-01-01

Tumorigenesis is a dynamic biological process that involves distinct cancer cell subpopulations proliferating at different rates and interconverting between them. In this paper we proposed a mathematical framework of population dynamics that considers both distinctive growth rates and intercellular transitions between cancer cell populations. Our mathematical framework showed that both growth and transition influence the ratio of cancer cell subpopulations but the latter is more significant. We derived the condition that different cancer cell types can maintain distinctive subpopulations and we also explain why there always exists a stable fixed ratio after cell sorting based on putative surface markers. The cell fraction ratio can be shifted by changing either the growth rates of the subpopulations (Darwinism selection) or by environment-instructed transitions (Lamarckism induction). This insight can help us to understand the dynamics of the heterogeneity of cancer cells and lead us to new strategies to overcome cancer drug resistance. PMID:25438251

Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis

PubMed Central

Tsujikawa, Takahiro; Kumar, Sushil; Borkar, Rohan N.; Azimi, Vahid; Thibault, Guillaume; Chang, Young Hwan; Balter, Ariel; Kawashima, Rie; Choe, Gina; Sauer, David; El Rassi, Edward; Clayburgh, Daniel R.; Kulesz-Martin, Molly F.; Lutz, Eric R.; Zheng, Lei; Jaffee, Elizabeth M.; Leyshock, Patrick; Margolin, Adam A.; Mori, Motomi; Gray, Joe W.; Flint, Paul W.; Coussens, Lisa M.

2017-01-01

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment. PMID:28380359

Reliability of High-Temperature Fixed-Point Installations over 8 Years

NASA Astrophysics Data System (ADS)

Elliott, C. J.; Ford, T.; Ongrai, O.; Pearce, J. V.

2017-12-01

At NPL, high-temperature metal-carbon eutectic fixed points have been set up for thermocouple calibration purposes since 2006, for realising reference temperatures above the highest point specified in the International Temperature Scale of 1990 for contact thermometer calibrations. Additionally, cells of the same design have been provided by NPL to other national measurement institutes (NMIs) and calibration laboratories over this period, creating traceable and ISO 17025 accredited facilities around the world for calibrating noble metal thermocouples at 1324 {°}C (Co-C) and 1492 {°}C (Pd-C). This paper shows collections of thermocouple calibration results obtained during use of the high-temperature fixed-point cells at NPL and, as further examples, the use of cells installed at CCPI Europe (UK) and NIMT (Thailand). The lifetime of the cells can now be shown to be in excess of 7 years, whether used on a weekly or monthly basis, and whether used in an NMI or industrial calibration laboratory.

Fixed-bed biosorption of cadmium using immobilized Scenedesmus obliquus CNW-N cells on loofa (Luffa cylindrica) sponge.

PubMed

Chen, Bor-Yann; Chen, Chun-Yen; Guo, Wan-Qian; Chang, Hao-Wei; Chen, Wen-Ming; Lee, Duu-Jong; Huang, Chieh-Chen; Ren, Nan-Qi; Chang, Jo-Shu

2014-05-01

A continuous fixed-bed biosorption process was established for cadmium (Cd) removal by Scenedesmus obliquus CNW-N (isolated from southern Taiwan) cells immobilized onto loofa sponge. This immobilized-cell biosorption process allows better recovery and reusability of the microalgal biomass. The growth of microalgae on the matrix support with appropriate nutrient supplementation could enhance the overall metal removal activity. Major operating parameters (e.g., feeding flow rate, cycle number of medium replacement, and particle diameter of the sponge) were studied for treatability evaluation. The most promising cell growth on the sponge support was obtained at a flow rate of 0.284 bed volume (BV)/min, sponge particle diameter of 1 cm, and with one cycle of medium replacement. The performance of fixed-bed biosorption (adsorption capacity of 38.4 mg, breakthrough time at 15.5 h) was achieved at a flow rate of 5 ml/min with an influent concentration of 7.5 mg Cd/l. Copyright © 2014 Elsevier Ltd. All rights reserved.

Functional nucleic acids as in vivo metabolite and ion biosensors.

PubMed

Alsaafin, Alaa; McKeague, Maureen

2017-08-15

Characterizing the role of metabolites, metals, and proteins is required to understand normal cell function, and ultimately, elucidate the mechanism of disease. Metabolite concentration and transformation results collected from cell lysates or fixed-cells conceal important dynamic information and differences between individual cells that often have profound functional consequences. Functional nucleic acid-based biosensors are emerging tools that are capable of monitoring ions and metabolites in cell populations or whole animals. Functional nucleic acids (FNAs) are a class of biomolecules that can exhibit either ligand binding or enzymatic activity. Unlike their protein analogues or the use of instrument-based analysis, FNA-based biosensors are capable of entering cells without disruption to the cellular environment and can report on the concentration, dynamics, and spatial localization of molecules in cells. Here, we review the types of FNAs that have been used as in vivo biosensors, and how FNAs can be coupled to transduction systems and delivered inside cells. We also provide examples from the literature that demonstrate their impact in practical applications. Finally, we comment on the critical limitations that need to be addressed to enable their use for single-cell dynamic tracking of metabolites and ions in vivo. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation.

PubMed

Kubota, E; McKenzie, D T; Dutton, R W; Swain, S L

1991-01-01

Antigen-unselected helper T-cell hybridomas (Th) which activate normal resting B cells to RNA synthesis and proliferation in the presence of concanavalin A (Con A) have been developed. The response is completely Th cell dependent, and not restricted by the haplotype of the B-cell major histocompatibility complex (MHC). Culture supernatants from the Con A-stimulated Th hybridomas contain interleukin-4 (IL-4) and IL-2, but undetectable level of IL-5. The supernatant alone, however, does not induce B-cell activation or proliferation. Although the Con A-mediated Th cell-dependent B-cell response occurs in an MHC-unrestricted manner, the response of resting B cells can be blocked by monoclonal Ia antibody specific for the surface class II molecules of the responding B cell. The response is also blocked by monoclonal antibody to L3T4. Significant activation and proliferation of resting B cells can also be triggered by glutaraldehyde-fixed Th hybridomas and Con A when exogenous IL-4 is added. The stimulation with fixed Th hybridomas plus IL-4 can be inhibited by monoclonal anti-L3T4 or anti-Ia. These results suggest that maximal B-cell activation requires a direct helper T cell-B cell interaction which depends on availability of Ia on the B cell and L3T4 on the T cell, even when Con A overcomes the requirement for MHC-restricted T-cell recognition. We suggest that this signal, in conjunction with T-cell produced lymphokine IL-4, is responsible for the activation and subsequent proliferation of the B cells which occurs following interaction with T cells.

How Polycomb-Mediated Cell Memory Deals With a Changing Environment: Variations in PcG complexes and proteins assortment convey plasticity to epigenetic regulation as a response to environment.

PubMed

Marasca, Federica; Bodega, Beatrice; Orlando, Valerio

2018-04-01

Cells and tissues are continuously exposed to a changing microenvironment, hence the necessity of a flexible modulation of gene expression that in complex organism have been achieved through specialized chromatin mechanisms. Chromatin-based cell memory enables cells to maintain their identity by fixing lineage specific transcriptional programs, ensuring their faithful transmission through cell division; in particular PcG-based memory system evolved to maintain the silenced state of developmental and cell cycle genes. In evolution the complexity of this system have increased, particularly in vertebrates, indicating combinatorial and dynamic properties of Polycomb proteins, in some cases even overflowing outside the cell nucleus. Therefore, their function may not be limited to the imposition of rigid states of genetic programs, but on the ability to recognize signals and allow plastic transcriptional changes in response to different stimuli. Here, we discuss the most novel PcG mediated memory functions in facing and responding to the challenges posed by a fluctuating environment. © 2018 The Authors. BioEssays Published by WILEY Periodicals, Inc.

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

PubMed Central

Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

2016-01-01

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule early senescence and defense-like reactions.

PubMed

Bourcy, Marie; Brocard, Lysiane; Pislariu, Catalina I; Cosson, Viviane; Mergaert, Peter; Tadege, Millon; Mysore, Kirankumar S; Udvardi, Michael K; Gourion, Benjamin; Ratet, Pascal

2013-03-01

Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. Nodule cells contain large numbers of bacteroids which are differentiated, nitrogen-fixing forms of the symbiotic bacteria. In the nodules, symbiotic plant cells home and maintain hundreds of viable bacteria. In order to better understand the molecular mechanism sustaining the phenomenon, we searched for new plant genes required for effective symbiosis. We used a combination of forward and reverse genetics approaches to identify a gene required for nitrogen fixation, and we used cell and molecular biology to characterize the mutant phenotype and to gain an insight into gene function. The symbiotic gene DNF2 encodes a putative phosphatidylinositol phospholipase C-like protein. Nodules formed by the mutant contain a zone of infected cells reduced to a few cell layers. In this zone, bacteria do not differentiate properly into bacteroids. Furthermore, mutant nodules senesce rapidly and exhibit defense-like reactions. This atypical phenotype amongst Fix(-) mutants unravels dnf2 as a new actor of bacteroid persistence inside symbiotic plant cells. © 2012 CNRS. New Phytologist © 2012 New Phytologist Trust.

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy.

PubMed

Gianoncelli, A; Vaccari, L; Kourousias, G; Cassese, D; Bedolla, D E; Kenig, S; Storici, P; Lazzarino, M; Kiskinova, M

2015-05-14

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

Effect of contact time and force on monocyte adhesion to vascular endothelium.

PubMed Central

Rinker, K D; Prabhakar, V; Truskey, G A

2001-01-01

In this study we examined whether monocytic cell attachment to vascular endothelium was affected by elevating shear stress at a constant shear rate. Contact time, which is inversely related to the shear rate, was fixed and viscosity elevated with dextran to increase the shear stress (and hence the net force on the cell) independently of shear rate. At a fixed contact time, tethering frequencies increased, rolling velocities decreased, and median arrest durations increased with increasing shear stress. Rolling and short arrests (< 0.2 s) were well fit by a single exponential consistent with adhesion via the formation of a single additional bond. The cell dissociation constant, k(off), increased when the shear stress was elevated at constant shear rate. Firmly adherent cells arresting for at least 0.2 s were well fit by a stochastic model involving dissociation from multiple bonds. Therefore, at a fixed contact time and increasing shear stress, bonds formed more frequently for rolling cells resulting in more short arrests, and more bonds formed for firmly arresting cells resulting in longer arrest durations. Possible mechanisms for this increased adhesion include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers. PMID:11259286

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy

PubMed Central

Gianoncelli, A.; Vaccari, L.; Kourousias, G.; Cassese, D.; Bedolla, D. E.; Kenig, S.; Storici, P.; Lazzarino, M.; Kiskinova, M.

2015-01-01

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies. PMID:25974639

10 CFR 603.300 - Difference between an expenditure-based and a fixed-support TIA.

Code of Federal Regulations, 2010 CFR

2010-01-01

... TECHNOLOGY INVESTMENT AGREEMENTS Requirements for Expenditure-Based and Fixed-Support Technology Investment... requirements in this subpart. The fundamental difference between an expenditure-based and a fixed-support TIA...

Automated Extraction of Formalin-Fixed, Paraffin-Embedded Tissue for High-Risk Human Papillomavirus Testing of Head and Neck Squamous Cell Carcinomas Using the Roche Cobas 4800 System.

PubMed

Kerr, Darcy A; Sweeney, Brenda; Arpin, Ronald N; Ring, Melissa; Pitman, Martha B; Wilbur, David C; Faquin, William C

2016-08-01

-Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. -To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. -Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. -Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. -Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

A dummy cell immersed boundary method for incompressible turbulence simulations over dirty geometries

NASA Astrophysics Data System (ADS)

Onishi, Keiji; Tsubokura, Makoto

2016-11-01

A methodology to eliminate the manual work required for correcting the surface imperfections of computer-aided-design (CAD) data, will be proposed. Such a technique is indispensable for CFD analysis of industrial applications involving complex geometries. The CAD geometry is degenerated into cell-oriented values based on Cartesian grid. This enables the parallel pre-processing as well as the ability to handle 'dirty' CAD data that has gaps, overlaps, or sharp edges without necessitating any fixes. An arbitrary boundary representation is used with a dummy-cell technique based on immersed boundary (IB) method. To model the IB, a forcing term is directly imposed at arbitrary ghost cells by linear interpolation of the momentum. The mass conservation is satisfied in the approximate domain that covers fluid region except the wall including cells. Attempts to Satisfy mass conservation in the wall containing cells leads to pressure oscillations near the IB. The consequence of this approximation will be discussed through fundamental study of an LES based channel flow simulation, and high Reynolds number flow around a sphere. And, an analysis comparing our results with wind tunnel experiments of flow around a full-vehicle geometry will also be presented.

47 CFR 90.1307 - Licensing.

Code of Federal Regulations, 2010 CFR

2010-10-01

... licenses will serve as a prerequisite for registering individual fixed and base stations. A licensee cannot operate a fixed or base station before registering it under its license and licensees must delete registrations for unused fixed and base stations. ...

Convergence of methods for coupling of microscopic and mesoscopic reaction-diffusion simulations

NASA Astrophysics Data System (ADS)

Flegg, Mark B.; Hellander, Stefan; Erban, Radek

2015-05-01

In this paper, three multiscale methods for coupling of mesoscopic (compartment-based) and microscopic (molecular-based) stochastic reaction-diffusion simulations are investigated. Two of the three methods that will be discussed in detail have been previously reported in the literature; the two-regime method (TRM) and the compartment-placement method (CPM). The third method that is introduced and analysed in this paper is called the ghost cell method (GCM), since it works by constructing a "ghost cell" in which molecules can disappear and jump into the compartment-based simulation. Presented is a comparison of sources of error. The convergent properties of this error are studied as the time step Δt (for updating the molecular-based part of the model) approaches zero. It is found that the error behaviour depends on another fundamental computational parameter h, the compartment size in the mesoscopic part of the model. Two important limiting cases, which appear in applications, are considered: Δt → 0 and h is fixed; Δt → 0 and h → 0 such that √{ Δt } / h is fixed. The error for previously developed approaches (the TRM and CPM) converges to zero only in the limiting case (ii), but not in case (i). It is shown that the error of the GCM converges in the limiting case (i). Thus the GCM is superior to previous coupling techniques if the mesoscopic description is much coarser than the microscopic part of the model.

Analysis of Pheochromocytoma (PC12) Membrane Potential under the Exposure to Millimeter-wave Radiation

NASA Astrophysics Data System (ADS)

Mizuno, M.; Hirata, A.; Kawase, K.; Otani, C.; Nagatsuma, T.

2004-08-01

Non-thermal effects of millimeter wave (MMW) on Pheochromocytoma (PC12) were studied by potential measurement with a voltage sensitive dye (DiBAC4(3)). Cells were irradiated at fixed frequencies of 30, 40, 60, 76GHz as well as sweeping frequency between 10 and 100 GHz by an MMW generator based on a uni-traveling-carrier photodiode (UTC-PD), the most widely tunable MMW source. However there were no significant changes in membrane potential between MMW-irradiated and control cells. The results suggest that MMW irradiation in the range from 10 to 100GHz appears to be safe for ordinary PC12 cells under non-thermal conditions.

Quantitation by flow microfluorometry of total cellular DNA in Acanthamoeba

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

Coulson, P.B.; Tyndall, R.

1978-01-01

The DNA content of five speciea of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24 to 48 hr. The trophozoites were harvested, fixed in 70% ethanol (acidified), pretreated with RNase, stained with propidium diiodide, and evaluated for DNA-bound fluorescence. All species tested had DNA values between 2.0 to 5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMFmore » may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.« less

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-10-02

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-11-01

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells

PubMed Central

Delfour, Christophe; Roger, Pascal; Bret, Caroline; Berthe, Marie-Laurence; Rochaix, Philippe; Kalfa, Nicolas; Raynaud, Pierre; Bibeau, Frédéric; Maudelonde, Thierry; Boulle, Nathalie

2006-01-01

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions. PMID:16645201

The response of single human cells to zero gravity

NASA Technical Reports Server (NTRS)

Montgomery, P. O., Jr.; Cook, J. E.; Reynolds, R. C.; Paul, J. S.; Hayflick, L.; Schulz, W. W.; Stock, D.; Kinzey, S.; Rogers, T.; Campbell, D.

1975-01-01

Twenty separate cultures of Wistar-38 human embryonic lung cells were exposed to a zero-gravity environment on Skylab for periods of time ranging from one to 59 days. Duplicate cultures were run concurrently as ground controls. Ten cultures were fixed on board the satellite during the first 12 days of flight. Growth curves, DNA microspectrophotometry, phase microscopy, and ultrastructural studies of the fixed cells revealed no effects of a zero-gravity environment on the ten cultures. Two cultures were photographed with phase time lapse cinematography during the first 27 days of flight. No differences were found in mitotic index, cell cycle, and migration between the flight and control cells. Eight cultures were returned to earth in an incubated state. Karyotyping and chromosome banding tests show no differences between the flight and control cells.

Wall shear stress fixed points in blood flow

NASA Astrophysics Data System (ADS)

Arzani, Amirhossein; Shadden, Shawn

2017-11-01

Patient-specific computational fluid dynamics produces large datasets, and wall shear stress (WSS) is one of the most important parameters due to its close connection with the biological processes at the wall. While some studies have investigated WSS vectorial features, the WSS fixed points have not received much attention. In this talk, we will discuss the importance of WSS fixed points from three viewpoints. First, we will review how WSS fixed points relate to the flow physics away from the wall. Second, we will discuss how certain types of WSS fixed points lead to high biochemical surface concentration in cardiovascular mass transport problems. Finally, we will introduce a new measure to track the exposure of endothelial cells to WSS fixed points.

Imaging C. elegans embryos using an epifluorescent microscope and open source software.

PubMed

Verbrugghe, Koen J C; Chan, Raymond C

2011-03-24

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples(1,2). Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage(3), thus providing an ideal experiment model for studying questions in cell biology(4,5)and development(6-9). C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis(10,11)) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis(12-15)). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters(16,17). These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo(18-21). In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

A three-dimensional numerical simulation of cell behavior in a flow chamber based on fluid-solid interaction.

PubMed

Bai, Long; Cui, Yuhong; Zhang, Yixia; Zhao, Na

2014-01-01

The mechanical behavior of blood cells in the vessels has a close relationship with the physical characteristics of the blood and the cells. In this paper, a numerical simulation method was proposed to understand a single-blood cell's behavior in the vessels based on fluid-solid interaction method, which was conducted under adaptive time step and fixed time step, respectively. The main programme was C++ codes, which called FLUENT and ANSYS software, and UDF and APDL acted as a messenger to connect FLUENT and ANSYS for exchanging data. The computing results show: (1) the blood cell moved towards the bottom of the flow chamber in the beginning due to the influence of gravity, then it began to jump up when reached a certain height rather than touching the bottom. It could move downwards again after jump up, the blood cell could keep this way of moving like dancing continuously in the vessels; (2) the blood cell was rolling and deforming all the time; the rotation had oscillatory changes and the deformation became conspicuously when the blood cell was dancing. This new simulation method and results can be widely used in the researches of cytology, blood, cells, etc.

In vitro FTIR microspectroscopy analysis of primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil: a new spectroscopic approach for studying the drug-cell interaction.

PubMed

Giorgini, Elisabetta; Sabbatini, Simona; Rocchetti, Romina; Notarstefano, Valentina; Rubini, Corrado; Conti, Carla; Orilisi, Giulia; Mitri, Elisa; Bedolla, Diana E; Vaccari, Lisa

2018-06-22

In the present study, human primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil were analyzed, for the first time, by in vitro FTIR Microspectroscopy (FTIRM), to improve the knowledge on the biochemical pathways activated by these two chemotherapy drugs. To date, most of the studies regarding FTIRM cellular analysis have been executed on fixed cells from immortalized cell lines. FTIRM analysis performed on primary tumor cells under controlled hydrated conditions provides more reliable information on the biochemical processes occurring in in vivo tumor cells. This spectroscopic analysis allows to get on the same sample and at the same time an overview of the composition and structure of the most remarkable cellular components. In vitro FTIRM analysis of primary oral squamous carcinoma cells evidenced a time-dependent drug-specific cellular response, also including apoptosis triggering. Furthermore, the univariate and multivariate analyses of IR data evidenced meaningful spectroscopic differences ascribable to alterations affecting cellular proteins, lipids and nucleic acids. These findings suggest for the two drugs different pathways and extents of cellular damage, not provided by conventional cell-based assays (MTT assay and image-based cytometry).

Mature phenotype in Hemerocallis plantlets fortuitously generated in vitro

NASA Technical Reports Server (NTRS)

Fitter, M. S.; Krikorian, A. D.

1985-01-01

Daylily plantlets generated on semi-solid media from morphogenetically competent cells or morphogenetically competent cells regenerated from protoplasts can give rise in aseptic culture to plantlets with a mature phenotype. The individual leaves of these plantlets open to the extreme base so that no encircling leaf sheath is present. This permits the overlapping bases and leaves to assume an open fan-like arrangement. The occurrence of fans correlates with exceptionally tightly sealed culture vessels and experiments to date suggest a gaseous component is associated with this change of growth form. It has not been possible to fix the mature growth mode, however, and new leaf growth assumes the more normal juvenile phenotype when the gaseous environment is altered by admitting or exposure to room air.

Fluorine-fixing efficiency on calcium-based briquette: pilot experiment, demonstration and promotion.

PubMed

Yang, Jiao-lan; Chen, Dong-qing; Li, Shu-min; Yue, Yin-ling; Jin, Xin; Zhao, Bing-cheng; Ying, Bo

2010-02-05

The fluorosis derived from coal burning is a very serious problem in China. By using fluorine-fixing technology during coal burning we are able to reduce the release of fluorides in coal at the source in order to reduce pollution to the surrounding environment by coal burning pollutants as well as decrease the intake and accumulating amounts of fluorine in the human body. The aim of this study was to conduct a pilot experiment on calcium-based fluorine-fixing material efficiency during coal burning to demonstrate and promote the technology based on laboratory research. A proper amount of calcium-based fluorine sorbent was added into high-fluorine coal to form briquettes so that the fluorine in high-fluorine coal can be fixed in coal slag and its release into atmosphere reduced. We determined figures on various components in briquettes and fluorine in coal slag as well as the concentrations of indoor air pollutants, including fluoride, sulfur dioxide and respirable particulate matter (RPM), and evaluated the fluorine-fixing efficiency of calcium-based fluorine sorbents and the levels of indoor air pollutants. Pilot experiments on fluorine-fixing efficiency during coal burning as well as its demonstration and promotion were carried out separately in Guiding and Longli Counties of Guizhou Province, two areas with coal burning fluorosis problems. If the calcium-based fluorine sorbent mixed coal was made into honeycomb briquettes the average fluorine-fixing ratio in the pilot experiment was 71.8%. If the burning calcium-based fluorine-fixing bitumite was made into a coalball, the average of fluorine-fixing ratio was 77.3%. The concentration of fluoride, sulfur dioxide and PM10 of indoor air were decreased significantly. There was a 10% increase in the cost of briquettes due to the addition of calcium-based fluorine sorbent. The preparation process of calcium-based fluorine-fixing briquette is simple yet highly flammable and it is applicable to regions with abundant bitumite coal. As a small scale application, villagers may make fluorine-fixing coalballs or briquettes by themselves, achieving the optimum fluorine-fixing efficiency and reducing indoor air pollutants providing environmental and social benefits.

Expression of human factor IX gene in murine plasma through lentiviral vector-infected haematopoietic stem cells.

PubMed

Chen, Haoming; Yao, Hengmei; Huang, Lu; Shen, Qi; Jia, William; Xue, Jinglun

2006-12-01

1. Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX (hFIX) gene, respectively, to infect HSC. 2. High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate-mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro. Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence-activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3. The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)-3, IL-6 and stem cell factor. 4. Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [(60)Co]-irradiated non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 +/- 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5. The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.

Epstein-Barr Virus, Human Papillomavirus and Mouse Mammary Tumour Virus as Multiple Viruses in Breast Cancer

PubMed Central

Glenn, Wendy K.; Heng, Benjamin; Delprado, Warick; Iacopetta, Barry; Whitaker, Noel J.; Lawson, James S.

2012-01-01

Background The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers. Materials and Methods All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc). Results EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk – EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples. Conclusions We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer. PMID:23183846

Epstein-Barr virus, human papillomavirus and mouse mammary tumour virus as multiple viruses in breast cancer.

PubMed

Glenn, Wendy K; Heng, Benjamin; Delprado, Warick; Iacopetta, Barry; Whitaker, Noel J; Lawson, James S

2012-01-01

The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers. All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc). EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk - EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples. We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer.

Comparison of two preparatory techniques for urine cytology.

PubMed Central

Dhundee, J; Rigby, H S

1990-01-01

Two methods of preparation of urine for cytology were compared retrospectively. In method 1 cells in the urine were fixed after the preparation of the smear; in method 2 the cells were fixed before smear preparation. Urine cytology reports were correlated with subsequent histological analysis. The specificities of urine cytology using both methods were high (99%). The sensitivity using method 1 was 87%; using method 2 it was 65%. This difference was significant. The cell preparation technique therefore significantly changes the sensitivity of urine cytology. Cellular fixation after smear preparation is preferable to smear preparation after fixation. PMID:2266176

Free flow cell electrophoresis using zwitterionic buffer

NASA Technical Reports Server (NTRS)

Rodkey, R. Scott

1990-01-01

Studies of a zwitterionic buffer formulated for cell electrophoresis were done using the McDonnell-Douglas Continuous Flow Electrophoresis System. Standard buffers were analyzed for their stability in the electrical field and the results showed that both buffers tested were inherently unstable. Further, titration studies showed that the standards buffers buffered poorly at the pH employed for electrophoresis. The zwitterionic buffer buffered well at its nominal pH and was shown to be stable in the electrical field. Comparative studies of the buffer with standard cell separation buffers using formalin fixed rabbit and goose red blood cells showed that the zwitterionic buffer gave better resolution of the fixed cells. Studies with viable hybridoma cells showed that buffer Q supported cell viability equal to Hank's Balanced Salt Solution and that hybridoma cells in different stages of the growth cycle demonstrated reproducible differences in electrophoretic mobility.

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

PubMed

Cunha, Nicolau B; Murad, André M; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Araújo, Ana Cláudia G; Lacorte, Cristiano; Aragão, Francisco J L; Covas, Dimas T; Fontes, Aparecida M; Souza, Gustavo H M F; Vianna, Giovanni R; Rech, Elíbio L

2011-08-01

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

NAVO MSRC Navigator. Fall 2006

DTIC Science & Technology

2006-01-01

UNIX Manual Pages: xdm (1x). 7. Buddenhagen, Oswald, “The KDM Handbook,” KDE Documentation, http://docs.kde.org/development/ en /kdebase/kdm/. 8... Linux Opteron cluster was recently determined through a series of simulations that employed both fixed and adaptive meshes. The fixed-mesh scalability...approximately eight in the total number of cells in the 3-D simulation. The fixed-mesh and AMR scalability results on the Linux Opteron cluster are

The Analysis of Fixed Final State Optimal Control in Bilinear System Applied to Bone Marrow by Cell-Cycle Specific (CCS) Chemotherapy

NASA Astrophysics Data System (ADS)

Rainarli, E.; E Dewi, K.

2017-04-01

The research conducted by Fister & Panetta shown an optimal control model of bone marrow cells against Cell Cycle Specific chemotherapy drugs. The model used was a bilinear system model. Fister & Panetta research has proved existence, uniqueness, and characteristics of optimal control (the chemotherapy effect). However, by using this model, the amount of bone marrow at the final time could achieve less than 50 percent from the amount of bone marrow before given treatment. This could harm patients because the lack of bone marrow cells made the number of leukocytes declining and patients will experience leukemia. This research would examine the optimal control of a bilinear system that applied to fixed final state. It will be used to determine the length of optimal time in administering chemotherapy and kept bone marrow cells on the allowed level at the same time. Before simulation conducted, this paper shows that the system could be controlled by using a theory of Lie Algebra. Afterward, it shows the characteristics of optimal control. Based on the simulation, it indicates that strong chemotherapy drug given in a short time frame is the most optimal condition to keep bone marrow cells spine on the allowed level but still could put playing an effective treatment. It gives preference of the weight of treatment for keeping bone marrow cells. The result of chemotherapy’s effect (u) is not able to reach the maximum value. On the other words, it needs to make adjustments of medicine’s dosage to satisfy the final treatment condition e.g. the number of bone marrow cells should be at the allowed level.

A simple method for determining polymeric IgA-containing immune complexes.

PubMed

Sancho, J; Egido, J; González, E

1983-06-10

A simplified assay to measure polymeric IgA-immune complexes in biological fluids is described. The assay is based upon the specific binding of a secretory component for polymeric IgA. In the first step, multimeric IgA (monomeric and polymeric) immune complexes are determined by the standard Raji cell assay. Secondly, labeled secretory component added to the assay is bound to polymeric IgA-immune complexes previously fixed to Raji cells, but not to monomeric IgA immune complexes. To avoid false positives due to possible complement-fixing IgM immune complexes, prior IgM immunoadsorption is performed. Using anti-IgM antiserum coupled to CNBr-activated Sepharose 4B this step is not time-consuming. Polymeric IgA has a low affinity constant and binds weakly to Raji cells, as Scatchard analysis of the data shows. Thus, polymeric IgA immune complexes do not bind to Raji cells directly through Fc receptors, but through complement breakdown products, as with IgG-immune complexes. Using this method, we have been successful in detecting specific polymeric-IgA immune complexes in patients with IgA nephropathy (Berger's disease) and alcoholic liver disease, as well as in normal subjects after meals of high protein content. This new, simple, rapid and reproducible assay might help to study the physiopathological role of polymeric IgA immune complexes in humans and animals.

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

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

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

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

An Electron-Deficient Building Block Based on the B←N Unit: An Electron Acceptor for All-Polymer Solar Cells.

PubMed

Dou, Chuandong; Long, Xiaojing; Ding, Zicheng; Xie, Zhiyuan; Liu, Jun; Wang, Lixiang

2016-01-22

A double B←N bridged bipyridyl (BNBP) is a novel electron-deficient building block for polymer electron acceptors in all-polymer solar cells. The B←N bridging units endow BNBP with fixed planar configuration and low-lying LUMO/HOMO energy levels. As a result, the polymer based on BNBP units (P-BNBP-T) exhibits high electron mobility, low-lying LUMO/HOMO energy levels, and strong absorbance in the visible region, which is desirable for polymer electron acceptors. Preliminary all-polymer solar cell (all-PSC) devices with P-BNBP-T as the electron acceptor and PTB7 as the electron donor exhibit a power conversion efficiency (PCE) of 3.38%, which is among the highest values of all-PSCs with PTB7 as the electron donor. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Physical-mechanical image of the cell surface on the base of AFM data in contact mode

NASA Astrophysics Data System (ADS)

Starodubtseva, M. N.; Starodubtsev, I. E.; Yegorenkov, N. I.; Kuzhel, N. S.; Konstantinova, E. E.; Chizhik, S. A.

2017-10-01

Physical and mechanical properties of the cell surface are well-known markers of a cell state. The complex of the parameters characterizing the cell surface properties, such as the elastic modulus (E), the parameters of adhesive (Fa), and friction (Ff) forces can be measured using atomic force microscope (AFM) in a contact mode and form namely the physical-mechanical image of the cell surface that is a fundamental element of the cell mechanical phenotype. The paper aims at forming the physical-mechanical images of the surface of two types of glutaraldehyde-fixed cancerous cells (human epithelial cells of larynx carcinoma, HEp-2c cells, and breast adenocarcinoma, MCF-7 cells) based on the data obtained by AFM in air and revealing the basic difference between them. The average values of friction, elastic and adhesive forces, and the roughness of lateral force maps, as well as dependence of the fractal dimension of lateral force maps on Z-scale factor have been studied. We have revealed that the response of microscale areas of the HEp-2c cell surface having numerous microvilli to external mechanical forces is less expressed and more homogeneous in comparison with the response of MCF-7 cell surface.

49 CFR 1562.25 - Fixed base operator requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 9 2011-10-01 2011-10-01 false Fixed base operator requirements. 1562.25 Section 1562.25 Transportation Other Regulations Relating to Transportation (Continued) TRANSPORTATION SECURITY... Operations § 1562.25 Fixed base operator requirements. (a) Security program. Each FBO must adopt and carry...

49 CFR 1562.25 - Fixed base operator requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 9 2013-10-01 2013-10-01 false Fixed base operator requirements. 1562.25 Section 1562.25 Transportation Other Regulations Relating to Transportation (Continued) TRANSPORTATION SECURITY... Operations § 1562.25 Fixed base operator requirements. (a) Security program. Each FBO must adopt and carry...

49 CFR 1562.25 - Fixed base operator requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 9 2012-10-01 2012-10-01 false Fixed base operator requirements. 1562.25 Section 1562.25 Transportation Other Regulations Relating to Transportation (Continued) TRANSPORTATION SECURITY... Operations § 1562.25 Fixed base operator requirements. (a) Security program. Each FBO must adopt and carry...

49 CFR 1562.25 - Fixed base operator requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 9 2010-10-01 2010-10-01 false Fixed base operator requirements. 1562.25 Section 1562.25 Transportation Other Regulations Relating to Transportation (Continued) TRANSPORTATION SECURITY... Operations § 1562.25 Fixed base operator requirements. (a) Security program. Each FBO must adopt and carry...

Directional reversals enable Myxococcus xanthus cells to produce collective one-dimensional streams during fruiting-body formation

PubMed Central

Thutupalli, Shashi; Sun, Mingzhai; Bunyak, Filiz; Palaniappan, Kannappan; Shaevitz, Joshua W.

2015-01-01

The formation of a collectively moving group benefits individuals within a population in a variety of ways. The surface-dwelling bacterium Myxococcus xanthus forms dynamic collective groups both to feed on prey and to aggregate during times of starvation. The latter behaviour, termed fruiting-body formation, involves a complex, coordinated series of density changes that ultimately lead to three-dimensional aggregates comprising hundreds of thousands of cells and spores. How a loose, two-dimensional sheet of motile cells produces a fixed aggregate has remained a mystery as current models of aggregation are either inconsistent with experimental data or ultimately predict unstable structures that do not remain fixed in space. Here, we use high-resolution microscopy and computer vision software to spatio-temporally track the motion of thousands of individuals during the initial stages of fruiting-body formation. We find that cells undergo a phase transition from exploratory flocking, in which unstable cell groups move rapidly and coherently over long distances, to a reversal-mediated localization into one-dimensional growing streams that are inherently stable in space. These observations identify a new phase of active collective behaviour and answer a long-standing open question in Myxococcus development by describing how motile cell groups can remain statistically fixed in a spatial location. PMID:26246416

Nano interface potential influences in CdTe quantum dots and biolabeling

NASA Astrophysics Data System (ADS)

Kanagasubbulakshmi, S.; Kadirvelu, K.

2018-05-01

Nano interface influences in physiochemical properties of quantum dots (QDs) are the challenging approach to tailor its surface functionalities. In this study, a set of polar and non-polar solvents were selected to analyze the influences in solvent-based dynamic radius and surface potential of QDs. From the nano interface chemistry of polar and non-polar solvents, an appropriate mechanism of precipitation and hydrophobic ligand exchange strategy were elucidated by correlating Henry's equation. Further, the in vitro cytotoxic potential and antimicrobial activity of QDs were assessed to perform biolabeling. From the observations, an appropriate dosage of QDs was fixed to label the animal ((RAW 264.7 cell lines) and bacterial cells (Escherichia coli) for effective cell attachment. Biolabeling was achieved by tailoring nano interface chemistry of QDs without additional support of biomolecules. Bacterial cell wall-based interaction of QDs was evaluated using SEM and EDAX analysis. Thus, provided clear insights into the nano interface chemistry in the development of highly photostable QDs will be helpful in biomedical applications.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

PubMed

Carro, Lorena; Pujic, Petar; Alloisio, Nicole; Fournier, Pascale; Boubakri, Hasna; Hay, Anne E; Poly, Franck; François, Philippe; Hocher, Valerie; Mergaert, Peter; Balmand, Severine; Rey, Marjolaine; Heddi, Abdelaziz; Normand, Philippe

2015-08-01

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

Evaluation of Pistacia lentiscus seed oil and phenolic compounds for in vitro antiproliferative effects against BHK21 cells.

PubMed

Mezni, Faten; Shili, Sarra; Ben Ali, Nejia; Larbi Khouja, Mohamed; Khaldi, Abdelhamid; Maaroufi, Abderrazak

2016-01-01

Within the global context of increasing cancer diseases, natural products are important in devising new drugs and providing unique ideas in cancer therapy. In Tunisian folk medicine, Pistacia lentiscus L. (Anacardiaceae) fixed oil is used for cancer treatment. This investigation studied, for the first time, the antiproliferative effect of Pistacia lentiscus fixed oil and its phenolic extract on BHK21 cancer cells. Oil was extracted from fruits harvested in northwest Tunisia and the phenolic fraction was obtained by mixing with methanol. The anti-proliferative activity of the two tested substances on BHK 21 cells were investigated in vitro using trypan blue assays. Cells were treated with different concentrations of P. lentiscus oil (0.009, 0.018, 0.036, and 0.09 g/mL) and the phenolic extract (0.007, 0.014, 0.03, and 0.07 g/mL) for 24, 48, and 72 h. The inhibitory effect of Pistacia lentiscus fixed oil increases with the increase in dose. The IC50 value was estimated at 0.029 g/mL. The percentage of cell viability was 42.46 ± 3.4% at a dose of 0.09 g/mL and was significantly lower than that of the untreated control (96.24 ± 2.5%, p<0.01). The phenolic extract demonstrated a dose- and time-dependent inhibitory effect on BHK21 cell growth. After 48 h of incubation, the IC50 value was estimated at 0.15 g/mL. The results demonstrated the potential of Pistacia lentiscus fixed oil in treating cancer, as it is used in traditional medicine.

Precision of Multiple Reaction Monitoring Mass Spectrometry Analysis of Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

2012-01-01

We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18–20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens. PMID:22530795

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel.

PubMed

Bolgioni, Amanda F; Vittoria, Marc A; Ganem, Neil J

2018-05-14

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 °C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Reproducibility of WC-C High-Temperature Fixed Point

NASA Astrophysics Data System (ADS)

Grigoryeva, I. A.; Khlevnoy, B. B.; Solodilov, M. V.

2017-05-01

Reproducibility of the tungsten carbide-carbon peritectic (WC-C) fixed point (3021 K) was investigated by comparing six WC-C blackbody-type cells with each other. All the cells were built at VNIIOFI and had the same design with a cell outer diameter of 24 mm and a cavity opening diameter of 3 mm. Four cells were built using tungsten powder supplied by Alfa Aesar, while the other two cells used powder from Zhuzhou KETE (China). The nominal purity of both suppliers was 99.999 %. All the cells were compared in the same furnace. A reference cell in the second furnace was used for monitoring the stability of a radiation thermometer. Melting temperatures (given by the point of inflection) of all six cells agreed within ±45 mK. The reproducibility, as a standard deviation of the measured temperatures, can be estimated as 35 mK. The Zhuzhou KETE cells showed a slightly lower temperature than the Alfa Aesar cells: by 32 mK on average, which is comparable with repeatability of the measurements.

Engineering of a synthetic quadrastable gene network to approach Waddington landscape and cell fate determination.

PubMed

Wu, Fuqing; Su, Ri-Qi; Lai, Ying-Cheng; Wang, Xiao

2017-04-11

The process of cell fate determination has been depicted intuitively as cells travelling and resting on a rugged landscape, which has been probed by various theoretical studies. However, few studies have experimentally demonstrated how underlying gene regulatory networks shape the landscape and hence orchestrate cellular decision-making in the presence of both signal and noise. Here we tested different topologies and verified a synthetic gene circuit with mutual inhibition and auto-activations to be quadrastable, which enables direct study of quadruple cell fate determination on an engineered landscape. We show that cells indeed gravitate towards local minima and signal inductions dictate cell fates through modulating the shape of the multistable landscape. Experiments, guided by model predictions, reveal that sequential inductions generate distinct cell fates by changing landscape in sequence and hence navigating cells to different final states. This work provides a synthetic biology framework to approach cell fate determination and suggests a landscape-based explanation of fixed induction sequences for targeted differentiation.

Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.

PubMed

Zhang, Fan; Wang, Zhuo-min; Liu, Hong-yu; Bai, Yun; Wei, Sen; Li, Ying; Wang, Min; Chen, Jun; Zhou, Qing-hua

2010-01-01

To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.

Impurity Correction Techniques Applied to Existing Doping Measurements of Impurities in Zinc

NASA Astrophysics Data System (ADS)

Pearce, J. V.; Sun, J. P.; Zhang, J. T.; Deng, X. L.

2017-01-01

Impurities represent the most significant source of uncertainty in most metal fixed points used for the realization of the International Temperature Scale of 1990 (ITS-90). There are a number of different methods for quantifying the effect of impurities on the freezing temperature of ITS-90 fixed points, many of which rely on an accurate knowledge of the liquidus slope in the limit of low concentration. A key method of determining the liquidus slope is to measure the freezing temperature of a fixed-point material as it is progressively doped with a known amount of impurity. Recently, a series of measurements of the freezing and melting temperature of `slim' Zn fixed-point cells doped with Ag, Fe, Ni, and Pb were presented. Here, additional measurements of the Zn-X system are presented using Ga as a dopant, and the data (Zn-Ag, Zn-Fe, Zn-Ni, Zn-Pb, and Zn-Ga) have been re-analyzed to demonstrate the use of a fitting method based on Scheil solidification which is applied to both melting and freezing curves. In addition, the utility of the Sum of Individual Estimates method is explored with these systems in the context of a recently enhanced database of liquidus slopes of impurities in Zn in the limit of low concentration.

A CpG-methylation-based assay to predict survival in clear cell renal cell carcinoma

PubMed Central

Wei, Jin-Huan; Haddad, Ahmed; Wu, Kai-Jie; Zhao, Hong-Wei; Kapur, Payal; Zhang, Zhi-Ling; Zhao, Liang-Yun; Chen, Zhen-Hua; Zhou, Yun-Yun; Zhou, Jian-Cheng; Wang, Bin; Yu, Yan-Hong; Cai, Mu-Yan; Xie, Dan; Liao, Bing; Li, Cai-Xia; Li, Pei-Xing; Wang, Zong-Ren; Zhou, Fang-Jian; Shi, Lei; Liu, Qing-Zuo; Gao, Zhen-Li; He, Da-Lin; Chen, Wei; Hsieh, Jer-Tsong; Li, Quan-Zhen; Margulis, Vitaly; Luo, Jun-Hang

2015-01-01

Clear cell renal cell carcinomas (ccRCCs) display divergent clinical behaviours. Molecular markers might improve risk stratification of ccRCC. Here we use, based on genome-wide CpG methylation profiling, a LASSO model to develop a five-CpG-based assay for ccRCC prognosis that can be used with formalin-fixed paraffin-embedded specimens. The five-CpG-based classifier was validated in three independent sets from China, United States and the Cancer Genome Atlas data set. The classifier predicts the overall survival of ccRCC patients (hazard ratio=2.96−4.82; P=3.9 × 10−6−2.2 × 10−9), independent of standard clinical prognostic factors. The five-CpG-based classifier successfully categorizes patients into high-risk and low-risk groups, with significant differences of clinical outcome in respective clinical stages and individual ‘stage, size, grade and necrosis' scores. Moreover, methylation at the five CpGs correlates with expression of five genes: PITX1, FOXE3, TWF2, EHBP1L1 and RIN1. Our five-CpG-based classifier is a practical and reliable prognostic tool for ccRCC that can add prognostic value to the staging system. PMID:26515236

SH2 Domain Histochemistry.

PubMed

Buhs, Sophia; Nollau, Peter

2017-01-01

Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues. So far, SH2 profiling has largely been applied for the analysis of protein extracts with the limitation that information on spatial distribution and intensity of tyrosine phosphorylation within a tissue is lost. Here, we describe a novel SH2 domain based strategy for differential characterization of the state of tyrosine phosphorylation in formaldehyde-fixed and paraffin-embedded tissues. This approach demonstrates that SH2 domains may serve as very valuable tools for the analysis of the differential state of tyrosine phosphorylation in primary tissues fixed and processed under conditions frequently applied by routine pathology laboratories.

The structure of plasmodesmata as revealed by plasmolysis, detergent extraction, and protease digestion

PubMed Central

1991-01-01

Plasmodesmata or intercellular bridges that connect plant cells are cylindrical channels approximately 40 nm in diameter. Running through the center of each is a dense rod, the desmotubule, that is connected to the endoplasmic reticulum of adjacent cells. Fern, Onoclea sensibilis, gametophytes were cut in half and the cut surfaces exposed to the detergent, Triton X 100, then fixed. Although the plasma membrane limiting the plasmodesma is solubilized partially or completely, the desmotubule remains intact. Alternatively, if the cut surface is exposed to papain, then fixed, the desmotubule disappears, but the plasma membrane limiting the plasmodesmata remains intact albeit swollen and irregular in profile. Gametophytes were plasmolyzed, and then fixed. As the cells retract from their cell walls they leave behind the plasmodesmata still inserted in the cell wall. They can break cleanly when the cell proper retracts or can pull away portions of the plasma membrane of the cell with them. Where the desmotubule remains intact, the plasmodesma retains its shape. These images and the results with detergents and proteases indicate that the desmotubule provides a cytoskeletal element for each plasmodesma, an element that not only stabilizes the whole structure, but also limits its size and porosity. It is likely to be composed in large part of protein. Suggestions are made as to why this structure has been selected for in evolution. PMID:1993740

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

PubMed Central

Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

2008-01-01

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. PMID:18456721

Genotoxicity analysis of two halonitromethanes, a novel group of disinfection by-products (DBPs), in human cells treated in vitro

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

Liviac, Danae; Creus, Amadeu; Marcos, Ricard

Halonitromethanes (HNMs) constitute an emerging class of disinfection by-products (DBPs) produced when chlorine and/or ozone are used for water treatment. The HNMs are structurally similar to halomethanes, but have a nitro-group in place of hydrogen bonded to the central carbon atom. Since little information exists on the genotoxic potential of HNMs, a study has been carried out with two HNM compounds, namely trichloronitromethane (TCNM) and bromonitromethane (BNM) by using human cells. Primary damage induction has been measured with the Comet assay, which is used to determine both the repair kinetics of the induced damage and the proportion of induced oxidativemore » damage. In addition, the fixed DNA damage has been evaluated by using the micronucleus (MN) assay. The results obtained indicate that both compounds are genotoxic, inducing high levels of DNA breaks in the Comet assay, and that this DNA damage repairs well over time. In addition, oxidized bases constitute a high proportion of DNA-induced damage (50-75%). Contrarily, no positive effects were observed in the frequency of micronucleus, which measures both clastogenic and aneugenic effects, neither using TK6 cells nor peripheral blood lymphocytes. This lack of fixed genetic damage would minimize the potential mutagenic risk associated with HNMs exposure.« less

Electrophoretic cell separation by means of microspheres

NASA Technical Reports Server (NTRS)

Smolka, A. J. K.; Nerren, B. H.; Margel, S.; Rembaum, A.

1979-01-01

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immunospecific labeling of target populations using microspheres.

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans.

PubMed

Parma, A E; Cerone, S I; Sansinanea, S A; Ghezzi, M

1992-10-01

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti-Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

Improved NLDAS-2 Noah-simulated Hydrometeorological Products with an Interim Run

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

Xia, Youlong; Peter-Lidard, Christa; Huang, Maoyi

2015-02-28

In NLDAS-2 Noah simulation, the NLDAS team introduced an intermediate fix suggested by Slater et al. (2007) and Livneh et al. (2010) to reduce large sublimation. The fix is used to constraint surface exchange coefficient (CH) using CH =CHoriginal x max (1.0-RiB/0.5, 0.05) when atmospheric boundary layer is stable. RiB is Richardson number. In NLDAS-2 Noah version, this fix was used for all stable cases including snow-free grid cells. In this study, we simply applied this fix to the grid cells in which both stable atmospheric boundary layer and snow exist simultaneously excluding the snow-free grid cells as we recognizemore » that the fix constraint in NLDAS-2 is too strong. We make a 31-year (1979-2009) Noah NLDAS-2 interim (NoahI) run. We use observed streamflow, evapotranspiration, land surface temperature, soil temperature, and ground heat flux to evaluate the results simulated from NoahI and make the reasonable comparison with those simulated from NLDAS-2 Noah (Xia et al., 2012). The results show that NoahI has the same performance as Noah does for snow water equivalent simulation. However, NoahI significantly improved the other hydrometeorological products’ simulation as described above when compared to Noah and the observations. This simple modification is being installed to the next Noah version. The hydrometeorological products simulated from NoahI will be staged on NCEP public server for the public in future.« less

Copper removal using a heavy-metal resistant microbial consortium in a fixed-bed reactor.

PubMed

Carpio, Isis E Mejias; Machado-Santelli, Glaucia; Sakata, Solange Kazumi; Ferreira Filho, Sidney Seckler; Rodrigues, Debora Frigi

2014-10-01

A heavy-metal resistant bacterial consortium was obtained from a contaminated river in São Paulo, Brazil and utilized for the design of a fixed-bed column for the removal of copper. Prior to the design of the fixed-bed bioreactor, the copper removal capacity by the live consortium and the effects of copper in the consortium biofilm formation were investigated. The Langmuir model indicated that the sorption capacity of the consortium for copper was 450.0 mg/g dry cells. The biosorption of copper into the microbial biomass was attributed to carboxyl and hydroxyl groups present in the microbial biomass. The effect of copper in planktonic cells to form biofilm under copper rich conditions was investigated with confocal microscopy. The results revealed that biofilm formed after 72 h exposure to copper presented a reduced thickness by 57% when compared to the control; however 84% of the total cells were still alive. The fixed-bed bioreactor was set up by growing the consortium biofilm on granular activated carbon (GAC) and analyzed for copper removal. The biofilm-GAC (BGAC) column retained 45% of the copper mass present in the influent, as opposed to 17% in the control column that contained GAC only. These findings suggest that native microbial communities in sites contaminated with heavy metals can be immobilized in fixed-bed bioreactors and used to treat metal contaminated water. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fixed drug eruption associated with intravenous contrast media: report in a woman receiving iohexol.

PubMed

Wright, Natalie A; Cohen, Philip R

2011-07-01

Fixed drug eruption, a medication-associated mucocutaneous reaction, rarely presents as a delayed adverse reaction to intravenous non-ionic contrast media. We describe a 57-year-old woman with a history of metastatic renal cell carcinoma who repeatedly developed a sharply demarcated, erythematous patch on her left breast after receiving the iodinated non-ionic contrast media iohexol for staging computed tomography scans. Recurrent fixed drug eruption may be avoided by using another contrast medium. Prophylactic treatment with systemic corticosteroids may prevent repeated fixed drug eruption if an alternative contrast agent cannot be used.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

PubMed

Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

2016-07-01

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Red blood cells aligning inside innovative liquid crystal cell

NASA Astrophysics Data System (ADS)

Likhomanova, S. V.; Kamanin, A. A.; Kamanina, N. V.

2017-11-01

Investigation results of red blood cells (human erythrocytes) aligning and fixing inside the liquid crystal (LC) cell have been presented in the present paper. LC cells have been modified through the improved nanostructured relief and LC sensitized with intermolecular charge transfer complex COANP-C70.

Fossilized glycolipids reveal past oceanic N2 fixation by heterocystous cyanobacteria

PubMed Central

Bauersachs, Thorsten; Speelman, Eveline N.; Hopmans, Ellen C.; Reichart, Gert-Jan; Schouten, Stefan; Damsté, Jaap S. Sinninghe

2010-01-01

N2-fixing cyanobacteria play an essential role in sustaining primary productivity in contemporary oceans and freshwater systems. However, the significance of N2-fixing cyanobacteria in past nitrogen cycling is difficult to establish as their preservation potential is relatively poor and specific biological markers are presently lacking. Heterocystous N2-fixing cyanobacteria synthesize unique long-chain glycolipids in the cell envelope covering the heterocyst cell to protect the oxygen-sensitive nitrogenase enzyme. We found that these heterocyst glycolipids are remarkably well preserved in (ancient) lacustrine and marine sediments, unambiguously indicating the (past) presence of N2-fixing heterocystous cyanobacteria. Analysis of Pleistocene sediments of the eastern Mediterranean Sea showed that heterocystous cyanobacteria, likely as epiphytes in symbiosis with planktonic diatoms, were particularly abundant during deposition of sapropels. Eocene Arctic Ocean sediments deposited at a time of large Azolla blooms contained glycolipids typical for heterocystous cyanobacteria presently living in symbiosis with the freshwater fern Azolla, indicating that this symbiosis already existed in that time. Our study thus suggests that heterocystous cyanobacteria played a major role in adding “new” fixed nitrogen to surface waters in past stratified oceans. PMID:20966349

Lattice Gas Model Based Optimization of Plasma-Surface Processes for GaN-Based Compound Growth

NASA Astrophysics Data System (ADS)

Nonokawa, Kiyohide; Suzuki, Takuma; Kitamori, Kazutaka; Sawada, Takayuki

2001-10-01

Progress of the epitaxial growth technique for GaN-based compounds makes these materials attractive for applications in high temperature/high-power electronic devices as well as in short-wavelength optoelectronic devices. For MBE growth of GaN epilayer, atomic nitrogen is usually supplied from ECR-plasma while atomic Ga is supplied from conventional K-cell. To grow high-quality epilayer, fundamental knowledge of the detailed atomic process, such as adsorption, surface migration, incorporation, desorption and so forth, is required. We have studied the influence of growth conditions on the flatness of the growth front surface and the growth rate using Monte Carlo simulation based on the lattice gas model. Under the fixed Ga flux condition, the lower the nitrogen flux and/or the higher the growth temperature, the better the flatness of the front surface at the sacrifice of the growth rate of the epilayer. When the nitrogen flux is increased, the growth rate reaches saturation value determined from the Ga flux. At a fixed growth temperature, increasing of nitrogen to Ga flux ratio results in rough surface owing to 3-dimensional island formation. Other characteristics of MBE-GaN growth using ECR-plasma can be well reproduced.

Optoelectrical modeling of solar cells based on c-Si/a-Si:H nanowire array: focus on the electrical transport in between the nanowires.

PubMed

Levtchenko, Alexandra; Le Gall, Sylvain; Lachaume, Raphaël; Michallon, Jérôme; Collin, Stéphane; Alvarez, José; Djebbour, Zakaria; Kleider, Jean-Paul

2018-06-22

By coupling optical and electrical modeling, we have investigated the photovoltaic performances of p-i-n radial nanowires array based on crystalline p-type silicon (c-Si) core/hydrogenated amorphous silicon (a-Si:H) shell. By varying either the doping concentration of the c-Si core, or back contact work function we can separate and highlight the contribution to the cell's performance of the nanowires themselves (the radial cell) from the interspace between the nanowires (the planar cell). We show that the build-in potential (V bi ) in the radial and planar cells strongly depends on the doping of c-Si core and the work function of the back contact respectively. Consequently, the solar cell's performance is degraded if either the doping concentration of the c-Si core, or/and the work function of the back contact is too low. By inserting a thin (p) a-Si:H layer between both core/absorber and back contact/absorber, the performance of the solar cell can be improved by partly fixing the V bi at both interfaces due to strong electrostatic screening effect. Depositing such a buffer layer playing the role of an electrostatic screen for charge carriers is a suggested way of enhancing the performance of solar cells based on radial p-i-n or n-i-p nanowire array.

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane.

PubMed

Taniyama, Toshiyuki; Tsuda, Natsumi; Sueda, Shinji

2018-06-15

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda).

PubMed

Beuerlein, Knut; Löhr, Sandra; Westermann, Bettina; Ruth, Peter; Schipp, Rudolf

2002-12-01

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.

10 CFR 603.300 - Difference between an expenditure-based and a fixed-support TIA.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 4 2013-01-01 2013-01-01 false Difference between an expenditure-based and a fixed-support TIA. 603.300 Section 603.300 Energy DEPARTMENT OF ENERGY (CONTINUED) ASSISTANCE REGULATIONS... Agreements § 603.300 Difference between an expenditure-based and a fixed-support TIA. The contracting officer...

Fixed-time synchronization of memristor-based BAM neural networks with time-varying discrete delay.

PubMed

Chen, Chuan; Li, Lixiang; Peng, Haipeng; Yang, Yixian

2017-12-01

This paper is devoted to studying the fixed-time synchronization of memristor-based BAM neural networks (MBAMNNs) with discrete delay. Fixed-time synchronization means that synchronization can be achieved in a fixed time for any initial values of the considered systems. In the light of the double-layer structure of MBAMNNs, we design two similar feedback controllers. Based on Lyapunov stability theories, several criteria are established to guarantee that the drive and response MBAMNNs can realize synchronization in a fixed time. In particular, by changing the parameters of controllers, this fixed time can be adjusted to some desired value in advance, irrespective of the initial values of MBAMNNs. Numerical simulations are included to validate the derived results. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fuel Cell Power Plant Initiative. Volume 2; Preliminary Design of a Fixed-Base LFP/SOFC Power System

NASA Technical Reports Server (NTRS)

Veyo, S.E.

1997-01-01

This report documents the preliminary design for a military fixed-base power system of 3 MWe nominal capacity using Westinghouse's tubular Solid Oxide Fuel Cell [SOFC] and Haldor Topsoe's logistic fuels processor [LFP]. The LFP provides to the fuel cell a methane rich sulfur free fuel stream derived from either DF-2 diesel fuel, or JP-8 turbine fuel. Fuel cells are electrochemical devices that directly convert the chemical energy contained in fuels such as hydrogen, natural gas, or coal gas into electricity at high efficiency with no intermediate heat engine or dynamo. The SOFC is distinguished from other fuel cell types by its solid state ceramic structure and its high operating temperature, nominally 1000'C. The SOFC pioneered by Westinghouse has a tubular geometry closed at one end. A power generation stack is formed by aggregating many cells in an ordered array. The Westinghouse stack design is distinguished from other fuel cell stacks by the complete absence of high integrity seals between cell elements, cells, and between stack and manifolds. Further, the reformer for natural gas [predominantly methane] and the stack are thermally and hydraulically integrated with no requirement for process water. The technical viability of combining the tubular SOFC and a logistic fuels processor was demonstrated at 27 kWe scale in a test program sponsored by the Advanced Research Projects Agency [ARPA) and carried out at the Southern California Edison's [SCE] Highgrove generating station near San Bernardino, California in 1994/95. The LFP was a breadboard design supplied by Haldor Topsoe, Inc. under subcontract to Westinghouse. The test program was completely successful. The LFP fueled the SOFC for 766 hours on JP-8 and 1555 hours of DF-2. In addition, the fuel cell operated for 3261 hours on pipeline natural gas. Over the 5582 hours of operation, the SOFC generated 118 MVVH of electricity with no perceptible degradation in performance. The LFP processed military specification JP-8 and DF-2 removing the sulfur and reforming these liquid fuels to a methane rich gaseous fuel. Results of this program are documented in a companion report titled 'Final Report-Solid Oxide Fuel Cell/ Logistic Fuels Processor 27 kWe Power System'.

Phosphate assimilation in Rhizobium (Sinorhizobium) meliloti: identification of a pit-like gene.

PubMed

Bardin, S D; Voegele, R T; Finan, T M

1998-08-01

Rhizobium meliloti mutants defective in the phoCDET-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix-). We have previously reported that two classes of second-site mutations can suppress the Fix- phenotype of phoCDET mutants to Fix+. Here we show that one of these suppressor loci (sfx1) contains two genes, orfA and pit, which appear to form an operon transcribed in the order orfA-pit. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the orfA transcription start site. This mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that the orfA-pit genes encode a Pi transport system that is expressed in wild-type cells grown with excess Pi but repressed in cells under conditions of Pi limitation. In phoCDET mutant cells, orfA-pit expression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increases orfA-pit expression compensating for the deficiencies in phosphate assimilation and symbiosis of the phoCDET mutants.

The identification of novel loci required for appropriate nodule development in Medicago truncatula.

PubMed

Domonkos, Agota; Horvath, Beatrix; Marsh, John F; Halasz, Gabor; Ayaydin, Ferhan; Oldroyd, Giles E D; Kalo, Peter

2013-10-11

The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules. Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed. The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

Pure moment testing for spinal biomechanics applications: fixed versus 3D floating ring cable-driven test designs.

PubMed

Tang, Jessica A; Scheer, Justin K; Ames, Christopher P; Buckley, Jenni M

2012-02-23

Pure moment testing has become a standard protocol for in vitro assessment of the effect of surgical techniques or devices on the bending rigidity of the spine. Of the methods used for pure moment testing, cable-driven set-ups are popular due to their low requirements and simple design. Fixed loading rings are traditionally used in conjunction with these cable-driven systems. However, the accuracy and validity of the loading conditions applied with fixed ring designs have raised some concern, and discrepancies have been found between intended and prescribed loading conditions for flexion-extension. This study extends this prior work to include lateral bending and axial torsion, and compares this fixed ring design with a novel "3D floating ring" design. A complete battery of multi-axial bending tests was conducted with both rings in multiple different configurations using an artificial lumbar spine. Applied moments were monitored and recorded by a multi-axial load cell at the base of the specimen. Results indicate that the fixed ring design deviates as much as 77% from intended moments and induces non-trivial shear forces (up to 18 N) when loaded to a non-destructive maximum of 4.5 Nm. The novel 3D floating ring design largely corrects the inherent errors in the fixed ring design by allowing additional directions of unconstrained motion and producing uniform loading conditions along the length of the specimen. In light of the results, it is suggested that the 3D floating ring set-up be used for future pure moment spine biomechanics applications using a cable-driven apparatus. Copyright © 2012 Elsevier Ltd. All rights reserved.

Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay.

PubMed Central

Sullender, W M; Anderson, L J; Anderson, K; Wertz, G W

1990-01-01

A new approach to respiratory syncytial (RS) virus subgroup determination was developed by using a simple nucleic acid filter hybridization technique. By this method, virus-infected cells are bound and fixed in a single step, and the viral RNA in the fixed-cell preparation is characterized directly by its ability to hybridize to cDNA probes specific for either the A or B subgroups of RS virus. The subgroup-specific probes were constructed from cDNA clones that corresponded to a portion of the extracellular domain of the RS virus G protein of either a subgroup B RS virus (8/60) or a subgroup A RS virus (A2). The cDNA probes were labeled with 32P and used to analyze RS virus isolates collected over a period of three decades. Replicate templates of infected cell preparations were hybridized with either the subgroup A or B probe. The subgroup assignments of 40 viruses tested by nucleic acid hybridization were in agreement with the results of subgroup determinations based on their reactivities with monoclonal antibodies, which previously has been the only method available for determining the subgroup classification of RS virus isolates. The nucleic acid hybridization assay has the advantage of providing broad-based discrimination of the two subgroups on the basis of nucleic acid homology, irrespective of minor antigenic differences that are detected in assays in which monoclonal antibodies are used. The nucleic acid hybridization technique provides a reliable method for RS virus subgroup characterization. Images PMID:2118548

Influence of sample preparation and identification of subcelluar structures in quantitative holographic phase contrast microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Schmidt, Lisa; Przibilla, Sabine; Rommel, Christina; Vollmer, Angelika; Ketelhut, Steffi; Schnekenburger, Jürgen; von Bally, Gert

2010-04-01

Digital holographic microscopy (DHM) provides label-free quantitative phase contrast with low demands on sample preparation. Nevertheless, for DHM measurements on fixed cells the mounting medium has to be considered while the phase contrast of living cells may be influenced by the used buffer solution. To quantify these effects, the maximum cell caused phase contrast and the visibility of the nucleoli were analyzed. A second aim of the study was to identify subcellular components in DHM phase contrast images. Therefore, comparative investigations using bright field imaging, DHM and fluorescence microscopy with 4',6- Diamidino-2-phenylindol (DAPI) staining were performed. DAPI-staining visualizes cell components containing DNA. The obtained results demonstrate exemplarily for two tumor cell lines that from DHM phase contrast images of fixed cells in phosphate buffer saline (PBS) cell thickness values are obtained which are comparable to living cells. Furthermore, it is shown that in many cases nucleus components can be identified only by DHM phase contrast.

Computer simulation of a cellular automata model for the immune response in a retrovirus system

NASA Astrophysics Data System (ADS)

Pandey, R. B.

1989-02-01

Immune response in a retrovirus system is modeled by a network of three binary cell elements to take into account some of the main functional features of T4 cells, T8 cells, and viruses. Two different intercell interactions are introduced, one of which leads to three fixed points while the other yields bistable fixed points oscillating between a healthy state and a sick state in a mean field treatment. Evolution of these cells is studied for quenched and annealed random interactions on a simple cubic lattice with a nearest neighbor interaction using inhomogenous cellular automata. Populations of T4 cells and viral cells oscillate together with damping (with constant amplitude) for annealed (quenched) interaction on increasing the value of mixing probability B from zero to a characteristic value B ca ( B cq). For higher B, the average number of T4 cells increases while that of the viral infected cells decreases monotonically on increasing B, suggesting a phase transition at B ca ( B cq).

Bioanalytical system for detection of cancer cells with photoluminescent ZnO nanorods

NASA Astrophysics Data System (ADS)

Viter, R.; Jekabsons, K.; Kalnina, Z.; Poletaev, N.; Hsu, S. H.; Riekstina, U.

2016-11-01

Using photoluminescent ZnO nanorods and carbohydrate marker SSEA-4, a novel cancer cell recognition system was developed. Immobilization of SSEA-4 antibodies (αSSEA-4) on ZnO nanorods was performed in buffer solution (pH = 7.1) over 2 h. The cancer cell line probes were fixed on the glass slide. One hundred microliters of ZnO-αSSEA-4 conjugates were deposited on the cell probe and exposed for 30 min. After washing photoluminescence spectra were recorded. Based on the developed methodology, ZnO-αSSEA-4 probes were tested on patient-derived breast and colorectal carcinoma cells. Our data clearly show that the carbohydrate SSEA-4 molecule is expressed on cancer cell lines and patient-derived cancer cells. Moreover, SSEA-4 targeted ZnO nanorods bind to the patient-derived cancer cells with high selectivity and the photoluminescence signal increased tremendously compared to the signal from the control samples. Furthermore, the photoluminescence intensity increase correlated with the extent of malignancy in the target cell population. A novel portable bioanalytical system, based on optical ZnO nanorods and fiber optic detection system was developed. We propose that carbohydrate SSEA-4 specific ZnO nanorods could be used for the development of cancer diagnostic biosensors and for targeted therapy.

Bandgap profiling in CIGS solar cells via valence electron energy-loss spectroscopy

NASA Astrophysics Data System (ADS)

Deitz, Julia I.; Karki, Shankar; Marsillac, Sylvain X.; Grassman, Tyler J.; McComb, David W.

2018-03-01

A robust, reproducible method for the extraction of relative bandgap trends from scanning transmission electron microscopy (STEM) based electron energy-loss spectroscopy (EELS) is described. The effectiveness of the approach is demonstrated by profiling the bandgap through a CuIn1-xGaxSe2 solar cell that possesses intentional Ga/(In + Ga) composition variation. The EELS-determined bandgap profile is compared to the nominal profile calculated from compositional data collected via STEM-based energy dispersive X-ray spectroscopy. The EELS based profile is found to closely track the calculated bandgap trends, with only a small, fixed offset difference. This method, which is particularly advantageous for relatively narrow bandgap materials and/or STEM systems with modest resolution capabilities (i.e., >100 meV), compromises absolute accuracy to provide a straightforward route for the correlation of local electronic structure trends with nanoscale chemical and physical structure/microstructure within semiconductor materials and devices.

New ways of looking at very small holes - using optical nanoscopy to visualize liver sinusoidal endothelial cell fenestrations

NASA Astrophysics Data System (ADS)

Øie, Cristina I.; Mönkemöller, Viola; Hübner, Wolfgang; Schüttpelz, Mark; Mao, Hong; Ahluwalia, Balpreet S.; Huser, Thomas R.; McCourt, Peter

2018-02-01

Super-resolution fluorescence microscopy, also known as nanoscopy, has provided us with a glimpse of future impacts on cell biology. Far-field optical nanoscopy allows, for the first time, the study of sub-cellular nanoscale biological structures in living cells, which in the past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy has particular utility in the study of "fenestrations" - phospholipid transmembrane nanopores of 50-150 nm in diameter through liver sinusoidal endothelial cells (LSECs) that facilitate the passage of plasma, but (usually) not blood cells, to and from the surrounding hepatocytes. Previously, these fenestrations were only discernible with EM, but now they can be visualized in fixed and living cells using structured illumination microscopy (SIM) and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy. Importantly, both methods use wet samples, avoiding dehydration artifacts. The use of nanoscopy can be extended to the in vitro study of fenestration dynamics, to address questions such as the following: are they actually dynamic structures, and how do they respond to endogenous and exogenous agents? A logical further extension of these methodologies to liver research (including the liver endothelium) will be their application to liver tissue sections from animal models with different pathological manifestations and ultimately to patient biopsies. This review will cover the current state of the art of the use of nanoscopy in the study of liver endothelium and the liver in general. Potential future applications in cell biology and the clinical implications will be discussed.

Induction of hapten-specific tolerance of human CD8+ urushiol (poison ivy)-reactive T lymphocytes.

PubMed

Kalish, R S; Wood, J A

1997-03-01

The interaction of CD28 with B7 molecules (CD80 or CD86) is an essential second signal for both the activation of CD4+ T cells through the T-cell receptor and the prevention of anergy. We studied the requirement of hapten-specific human CD8+ cells for CD28 co-stimulation in recognition of hapten, and anergy induction. Urushiol, the immunogenic hapten of poison ivy (Toxicodendron radicans), elicits a predominantly CD8+ T-cell response. Autologous PBMC were pre-incubated with urushiol prior to fixation by paraformaldehyde. Fixed antigen-presenting cells were unable to present urushiol to human CD8+ urushiol-specific T cells. Addition of anti-CD28, however, overcame this antigen-presenting defect, enabling CD8+ cells to proliferate. Fixation of antigen-presenting cells prevents upregulation of B7, and addition of anti-CD28 substitutes for this signal. Proliferation of CD8+ T cells in response to urushiol was blocked by CTLA4Ig, a recombinant fusion protein that blocks CD28/B7 interactions. Preincubation of urushiol-specific CD8+ cells with fixed PBMC + urushiol for 7 d induced anergy. Anergic CD8+ cells were viable and able to proliferate in response to IL-2, but not in response to urushiol. Induction of anergy required the presence of urushiol, and pre-incubation with irradiated PBMC + urushiol did not have this effect. It is proposed that anergy was induced by presentation of urushiol by fixed PBMC, in the absence of adequate co-stimulation signals. Induction of anergy by blocking of co-stimulation could potentially induce clinical hyposensitization to haptens.

Heparin conjugated quantum dots for in vitro imaging applications.

PubMed

Maguire, Ciaran Manus; Mahfoud, Omar Kazem; Rakovich, Tatsiana; Gerard, Valerie Anne; Prina-Mello, Adriele; Gun'ko, Yurii; Volkov, Yuri

2014-11-01

In this work heparin-gelatine multi-layered cadmium telluride quantum dots (QDgel/hep) were synthesised using a novel 'one-pot' method. The QDs produced were characterised using various spectroscopic and physiochemical techniques. Suitable QDs were then selected and compared to thioglycolic acid stabilised quantum dots (QDTGA) and gelatine coated quantum dots (QDgel) for utilisation in in vitro imaging experiments on live and fixed permeabilised THP-1, A549 and Caco-2 cell lines. Exposure of live THP-1 cells to QDgel/hep resulted in localisation of the QDs to the nucleus of the cells. QDgel/hep show affinity for the nuclear compartment of fixed permeabilised THP-1 and A549 cells but remain confined to cytoplasm of fixed permeabilised Caco-2 cells. It is postulated that heparin binding to the CD11b receptor facilitates the internalisation of the QDs into the nucleus of THP-1 cells. In addition, the heparin layer may reduce the unfavourable thrombogenic nature of quantum dots observed in vivo. In this study, heparin conjugated quantum dots were found to have superior imaging properties compared to its native counterparts. The authors postulate that heparin binding to the CD11b receptor facilitates QD internalization to the nucleus, and the heparin layer may reduce the in vivo thrombogenic properties of quantum dots. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein gradients in single cells induced by their coupling to "morphogen"-like diffusion

NASA Astrophysics Data System (ADS)

Nandi, Saroj Kumar; Safran, Sam A.

2018-05-01

One of the many ways cells transmit information within their volume is through steady spatial gradients of different proteins. However, the mechanism through which proteins without any sources or sinks form such single-cell gradients is not yet fully understood. One of the models for such gradient formation, based on differential diffusion, is limited to proteins with large ratios of their diffusion constants or to specific protein-large molecule interactions. We introduce a novel mechanism for gradient formation via the coupling of the proteins within a single cell with a molecule, that we call a "pronogen," whose action is similar to that of morphogens in multi-cell assemblies; the pronogen is produced with a fixed flux at one side of the cell. This coupling results in an effectively non-linear diffusion degradation model for the pronogen dynamics within the cell, which leads to a steady-state gradient of the protein concentration. We use stability analysis to show that these gradients are linearly stable with respect to perturbations.

Regulatory Response to Carbon Starvation in Caulobacter crescentus

PubMed Central

Britos, Leticia; Abeliuk, Eduardo; Taverner, Thomas; Lipton, Mary; McAdams, Harley; Shapiro, Lucy

2011-01-01

Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells. PMID:21494595

RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

PubMed

Kimura, Yurika; Kubo, Sachiho; Koda, Hiroko; Shigemoto, Kazuhiro; Sawabe, Motoji; Kitamura, Ken

2013-08-01

Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

NASA Astrophysics Data System (ADS)

Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.

2015-03-01

Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

Inactivation of infectious virus and serological detection of virus antigen in Rift Valley fever virus-exposed mosquitoes fixed with paraformaldehyde.

PubMed

Kading, Rebekah; Crabtree, Mary; Miller, Barry

2013-04-01

Formaldehyde is routinely used to fix tissues in preparation for pathology studies, however concerns remain that treatment of tissues with cellular fixatives may not entirely inactivate infectious virus particles. This concern is of particular regulatory importance for research involving viruses that are classified as select agents such as Rift Valley fever virus (RVFV). Therefore, the specific aims of this study were to (1) assay RVFV-exposed Aedes aegypti mosquitoes fixed in 4% paraformaldehyde for the presence of infectious RVFV particles at various time points following infection and (2) demonstrate the utility of immunofluorescence assay (IFA) for the detection of RVFV antigen in various tissues of paraformaldehyde-fixed mosquitoes. Mosquitoes were administered an infectious blood meal containing one of two strains of RVFV, harvested at various time points following infection, intrathoracically inoculated with 4% paraformaldehyde, and fixed overnight at 4°C. The infection status of a subset of mosquitoes was verified by IFA on leg tissues prior to fixation, and infectivity of RVFV in fixed mosquito carcasses was determined by Vero cell plaque assay. Paraformaldehyde-fixed mosquitoes harvested 14 days post infection were also paraffin-embedded and sectioned for detection of RVFV antigen to particular tissues by IFA. None of the RVFV-exposed mosquitoes tested by Vero cell plaque assay contained infectious RVFV after fixation. Furthermore, incubation of mosquito sections with trypsin prior to antibody staining is recommended for optimal visualization of RVFV antigen in infected mosquito tissues by IFA. Published by Elsevier B.V.

NASA Solar Array Demonstrates Commercial Potential

NASA Technical Reports Server (NTRS)

Creech, Gray

2006-01-01

A state-of-the-art solar-panel array demonstration site at NASA's Dryden Flight Research Center provides a unique opportunity for studying the latest in high-efficiency solar photovoltaic cells. This five-kilowatt solar-array site (see Figure 1) is a technology-transfer and commercialization success for NASA. Among the solar cells at this site are cells of a type that was developed in Dryden Flight Research Center s Environmental Research Aircraft and Sensor Technology (ERAST) program for use in NASA s Helios solar-powered airplane. This cell type, now denoted as A-300, has since been transferred to SunPower Corporation of Sunnyvale, California, enabling mass production of the cells for the commercial market. High efficiency separates these advanced cells from typical previously commercially available solar cells: Whereas typical previously commercially available cells are 12 to 15 percent efficient at converting sunlight to electricity, these advanced cells exhibit efficiencies approaching 23 percent. The increase in efficiency is due largely to the routing of electrical connections behind the cells (see Figure 2). This approach to increasing efficiency originated as a solution to the problem of maximizing the degree of utilization of the limited space available atop the wing of the Helios airplane. In retrospect, the solar cells in use at this site could be used on Helios, but the best cells otherwise commercially available could not be so used, because of their lower efficiencies. Historically, solar cells have been fabricated by use of methods that are common in the semiconductor industry. One of these methods includes the use of photolithography to define the rear electrical-contact features - diffusions, contact openings, and fingers. SunPower uses these methods to produce the advanced cells. To reduce fabrication costs, SunPower continues to explore new methods to define the rear electrical-contact features. The equipment at the demonstration site includes two fixed-angle solar arrays and one single-axis Sun-tracking array. One of the fixed arrays contains typical less-efficient commercial solar cells and is being used as a baseline for comparison of the other fixed array, which contains the advanced cells. The Sun-tracking array tilts to follow the Sun, using an advanced, real-time tracking device rather than customary pre-programmed mechanisms. Part of the purpose served by the demonstration is to enable determination of any potential advantage of a tracking array over a fixed array. The arrays are monitored remotely on a computer that displays pertinent information regarding the functioning of the arrays.

Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses

PubMed Central

Mariani, Sara; Tondat, Fabrizio; Pacchioni, Donatella; Molinaro, Luca; Barreca, Antonella; Macrì, Luigia; Chiusa, Luigi; di Celle, Paola Francia; Cassoni, Paola; Sapino, Anna

2015-01-01

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity. PMID:25844806

[Modification of Bowie's method of demonstrating specific granules in cells of the human renal juxtaglomerular apparatus fixed in neutral formalin].

PubMed

Orduian, S L

1976-04-01

A modification of Bowie's method for detection of specific granules of the juxtaglomerular apparatus of the human kidneys, fixed in 10% neutral formalin, is suggested. In order to achieve better staining, sections of material fixed in formalin are additionally treated with Helly's liquid and, following the removal of sublimate deposit, with a 2.5% solution of potassium bichromate. After this the sections are stained by Bowie's method in accordance with Pitcock and Hartroft's prescription.

Tempo-spatially resolved cellular dynamics of human immunodeficiency virus transacting activator of transcription (Tat) peptide-modified nanocargos in living cells

NASA Astrophysics Data System (ADS)

Wei, Lin; Yang, Qiaoyu; Xiao, Lehui

2014-08-01

Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery. Electronic supplementary information (ESI) available: Experimental section and additional supporting results as noted in the text. See DOI: 10.1039/c4nr02732a

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

Fixed Base Modal Survey of the MPCV Orion European Service Module Structural Test Article

NASA Technical Reports Server (NTRS)

Winkel, James P.; Akers, J. C.; Suarez, Vicente J.; Staab, Lucas D.; Napolitano, Kevin L.

2017-01-01

Recently, the MPCV Orion European Service Module Structural Test Article (E-STA) underwent sine vibration testing using the multi-axis shaker system at NASA GRC Plum Brook Station Mechanical Vibration Facility (MVF). An innovative approach using measured constraint shapes at the interface of E-STA to the MVF allowed high-quality fixed base modal parameters of the E-STA to be extracted, which have been used to update the E-STA finite element model (FEM), without the need for a traditional fixed base modal survey. This innovative approach provided considerable program cost and test schedule savings. This paper documents this modal survey, which includes the modal pretest analysis sensor selection, the fixed base methodology using measured constraint shapes as virtual references and measured frequency response functions, and post-survey comparison between measured and analysis fixed base modal parameters.

Microrobots for in vitro fertilization applications.

PubMed

Boukallel, M; Gauthier, M; Piat, E; Abadie, J; Roux, C

2004-05-01

The Micromanipulation and Micro-actuation Research Group at the LAB has activities related to biological and surgical applications. Concerning cells micromanipulation, our laboratory works in collaboration with the research team "Genetic and Reproduction" of the Besançon's hospital (France). The global final objective is the development of an automatic intra cytoplasmic sperm injection (ICSI) device in order to improve performances and ergonomics of current devices. In the future this new device will contain various modules: module for removal of cumulus cells, modules for characterization of oocytes, microinjection module, cells transport system. The first subsystem developed is a new single cell transport system. It consists in a so-called micropusher which pushes single cells without having contact with the external environment. This micropusher is a ferromagnetic particle (from 400 x 400 x 20 microm3 to 100 x 100 x 5 microm3) which follows the movement of a permanent magnet located under the biological medium. A 2D micro-positioning table moves this magnet under the glass slide. The pusher and cells positions are measured through an optical microscope with a CCD camera located above the biological medium. The second subsystem is developed to measure oocytes mechanical stiffness in order to sort them. We have then developed a micro/nano-force sensor based on the diamagnetic levitation principle: a glass tip end-effector (with 20 microm in diameter) is fixed on the equipment which is in levitation (0.5 mm in diameter, 100 mm in length). When a force is applied to the levitated glass tip, it moves to a new equilibrium position. Thanks to themeasurement of this displacement, the applied force can be measured. Since there is no contact and friction between the levitated tip and the fixed part, the resolution of this sensor is very high (10 nN).

Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

PubMed

Fernandes, Cláudia P H; Seixas, Fabiana K; Coutinho, Mariana L; Vasconcellos, Flávia A; Seyffert, Núbia; Croda, Julio; McBride, Alan J; Ko, Albert I; Dellagostin, Odir A; Aleixo, José A G

2007-02-01

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.

Long-Term Stability of Human Genomic and Human Papillomavirus DNA Stored in BD SurePath and Hologic PreservCyt Liquid-Based Cytology Media

PubMed Central

Agreda, Patricia M.; Beitman, Gerard H.; Gutierrez, Erin C.; Harris, James M.; Koch, Kristopher R.; LaViers, William D.; Leitch, Sharon V.; Maus, Courtney E.; McMillian, Ray A.; Nussbaumer, William A.; Palmer, Marcus L. R.; Porter, Michael J.; Richart, Gregory A.; Schwab, Ryan J.

2013-01-01

We evaluated the effect of storage at 2 to 8°C on the stability of human genomic and human papillomavirus (HPV) DNA stored in BD SurePath and Hologic PreservCyt liquid-based cytology media. DNA retained the ability to be extracted and PCR amplified for more than 2.5 years in both medium types. Prior inability to detect DNA in archived specimens may have been due to failure of the extraction method to isolate DNA from fixed cells. PMID:23678069

Analysis of Noise Mechanisms in Cell-Size Control.

PubMed

Modi, Saurabh; Vargas-Garcia, Cesar Augusto; Ghusinga, Khem Raj; Singh, Abhyudai

2017-06-06

At the single-cell level, noise arises from multiple sources, such as inherent stochasticity of biomolecular processes, random partitioning of resources at division, and fluctuations in cellular growth rates. How these diverse noise mechanisms combine to drive variations in cell size within an isoclonal population is not well understood. Here, we investigate the contributions of different noise sources in well-known paradigms of cell-size control, such as adder (division occurs after adding a fixed size from birth), sizer (division occurs after reaching a size threshold), and timer (division occurs after a fixed time from birth). Analysis reveals that variation in cell size is most sensitive to errors in partitioning of volume among daughter cells, and not surprisingly, this process is well regulated among microbes. Moreover, depending on the dominant noise mechanism, different size-control strategies (or a combination of them) provide efficient buffering of size variations. We further explore mixer models of size control, where a timer phase precedes/follows an adder, as has been proposed in Caulobacter crescentus. Although mixing a timer and an adder can sometimes attenuate size variations, it invariably leads to higher-order moments growing unboundedly over time. This results in a power-law distribution for the cell size, with an exponent that depends inversely on the noise in the timer phase. Consistent with theory, we find evidence of power-law statistics in the tail of C. crescentus cell-size distribution, although there is a discrepancy between the observed power-law exponent and that predicted from the noise parameters. The discrepancy, however, is removed after data reveal that the size added by individual newborns in the adder phase itself exhibits power-law statistics. Taken together, this study provides key insights into the role of noise mechanisms in size homeostasis, and suggests an inextricable link between timer-based models of size control and heavy-tailed cell-size distributions. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Imaging in focus: Imaging the dynamics of endocytosis.

PubMed

Rosendale, Morgane; Perrais, David

2017-12-01

Endocytosis, the formation of membrane vesicles from the plasma membrane, is an essential feature of eukaryotic cell biology. Intense research effort has been dedicated to developing methods that can detect endocytosis events with the highest resolution. We have classified these methods into four families. They exploit the physical properties of endocytosis, namely: 1. Distinguishing extracellular from internalised cargo in fixed samples, 2. Monitoring endosomal acidification, 3. Measuring the turnover of endocytic zones and 4. Detecting vesicle scission. The last three families, all based on fluorescence imaging, are used to study endocytosis in living cells. We discuss the advantages and limitations of these methods and conclude on the future developments required to tackle the upcoming challenges in this fundamental field of cell biology. Copyright © 2017. Published by Elsevier Ltd.

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

PubMed Central

Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

2014-01-01

Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

Self-calibration of a W/Re thermocouple using a miniature Ru-C (1954 °C) eutectic cell

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

Ongrai, O.; University of Surrey, Guildford, Surrey; National Institute of Metrology, Klong 5, Klong Luang, Pathumthani

2013-09-11

Previous successful investigations of miniature cobalt-carbon (Co-C, 1324 °C) and palladium-carbon (Pd-C, 1492 °C) high temperature fixed-point cells for thermocouple self-calibration have been reported [1-2]. In the present work, we describe a series of measurements of a miniature ruthenium-carbon (Ru-C) eutectic cell (melting point 1954 °C) to evaluate the repeatability and stability of a W/Re thermocouple (type C) by means of in-situ calibration. A miniature Ru-C eutectic fixed-point cell with outside diameter 14 mm and length 30 mm was fabricated to be used as a self-calibrating device. The performance of the miniature Ru-C cell and the type C thermocouple ismore » presented, including characterization of the stability, repeatability, thermal environment influence, ITS-90 temperature realization and measurement uncertainty.« less

Clinical value of miR-198-5p in lung squamous cell carcinoma assessed using microarray and RT-qPCR.

PubMed

Liang, Yue-Ya; Huang, Jia-Cheng; Tang, Rui-Xue; Chen, Wen-Jie; Chen, Peng; Cen, Wei-Luan; Shi, Ke; Gao, Li; Gao, Xiang; Liu, An-Gui; Peng, Xiao-Tong; Chen, Gang; Huang, Su-Ning; Fang, Ye-Ying; Gu, Yong-Yao

2018-02-02

To examine the clinical value of miR-198-5p in lung squamous cell carcinoma (LUSC). Gene Expression Omnibus (GEO) microarray datasets were used to explore the miR-198-5p expression and its diagnostic value in LUSC. Real-time reverse transcription quantitative polymerase chain reaction was used to evaluate the expression of miR-198-5p in 23 formalin-fixed, paraffin-embedded (FFPE) LUSC tissues and corresponding non-cancerous tissues. The correlation between miR-198-5p expression and clinic pathological features was assessed. Meanwhile, putative target messenger RNAs of miR-198-5p were identified based on the analysis of differentially expressed genes in the Cancer Genome Atlas (TCGA) and 12 miRNA prediction tools. Subsequently, the putative target genes were sent to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. MiR-198-5p was low expressed in LUSC tissues. The combined standard mean difference (SMD) values of miR-198-5p expression based on GEO datasets were - 0.30 (95% confidence interval (CI) - 0.54, - 0.06) and - 0.39 (95% CI - 0.83, 0.05) using fixed effect model and random effect model, respectively. The sensitivity and specificity were not sufficiently high, as the area under the curve (AUC) was 0.7749 (Q* = 0.7143) based on summarized receiver operating characteristic (SROC) curves constructed using GEO datasets. Based on the in-house RT-qPCR, miR-198-5p expression was 4.3826 ± 1.7660 in LUSC tissues and 4.4522 ± 1.8263 in adjacent normal tissues (P = 0.885). The expression of miR-198-5p was significantly higher in patients with early TNM stages (I-II) than that in cases with advanced TNM stages (III-IV) (5.4400 ± 1.5277 vs 3.5690 ± 1.5228, P = 0.008). Continuous variable-based meta-analysis of GEO and PCR data displayed the SMD values of - 0.26 (95% CI - 0.48, - 0.04) and - 0.34 (95% CI - 0.71, 0.04) based on fixed and random effect models, respectively. As for the diagnostic value of miR-198-5p, the AUC based on the SROC curve using GEO and PCR data was 0.7351 (Q* = 0.6812). In total, 542 genes were identified as the targets of miR-198-5p. The most enriched Gene Ontology terms were epidermis development among biological processes, cell junction among cellular components, and protein dimerization activity among molecule functions. The pathway of non-small cell lung cancer was the most significant pathway identified using Kyoto Encyclopedia of Genes and Genomes analysis. The expression of miR-198-5p is related to the TNM stage. Thus, miR-198-5p might play an important role via its target genes in LUSC.

Procedures for Behavioral Experiments in Head-Fixed Mice

PubMed Central

Guo, Zengcai V.; Hires, S. Andrew; Li, Nuo; O'Connor, Daniel H.; Komiyama, Takaki; Ophir, Eran; Huber, Daniel; Bonardi, Claudia; Morandell, Karin; Gutnisky, Diego; Peron, Simon; Xu, Ning-long; Cox, James; Svoboda, Karel

2014-01-01

The mouse is an increasingly prominent model for the analysis of mammalian neuronal circuits. Neural circuits ultimately have to be probed during behaviors that engage the circuits. Linking circuit dynamics to behavior requires precise control of sensory stimuli and measurement of body movements. Head-fixation has been used for behavioral research, particularly in non-human primates, to facilitate precise stimulus control, behavioral monitoring and neural recording. However, choice-based, perceptual decision tasks by head-fixed mice have only recently been introduced. Training mice relies on motivating mice using water restriction. Here we describe procedures for head-fixation, water restriction and behavioral training for head-fixed mice, with a focus on active, whisker-based tactile behaviors. In these experiments mice had restricted access to water (typically 1 ml/day). After ten days of water restriction, body weight stabilized at approximately 80% of initial weight. At that point mice were trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. In some cases mice learned to discriminate sensory stimuli in a few trials within the first behavioral session. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking). Mice performed a variety of perceptual decision tasks with high performance for hundreds of trials per behavioral session. Up to four months of continuous water restriction showed no adverse health effects. Behavioral performance correlated with the degree of water restriction, supporting the importance of controlling access to water. These behavioral paradigms can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings. PMID:24520413

Malaria Diagnosis Using a Mobile Phone Polarized Microscope

NASA Astrophysics Data System (ADS)

Pirnstill, Casey W.; Coté, Gerard L.

2015-08-01

Malaria remains a major global health burden, and new methods for low-cost, high-sensitivity, diagnosis are essential, particularly in remote areas with low-resource around the world. In this paper, a cost effective, optical cell-phone based transmission polarized light microscope system is presented for imaging the malaria pigment known as hemozoin. It can be difficult to determine the presence of the pigment from background and other artifacts, even for skilled microscopy technicians. The pigment is much easier to observe using polarized light microscopy. However, implementation of polarized light microscopy lacks widespread adoption because the existing commercial devices have complicated designs, require sophisticated maintenance, tend to be bulky, can be expensive, and would require re-training for existing microscopy technicians. To this end, a high fidelity and high optical resolution cell-phone based polarized light microscopy system is presented which is comparable to larger bench-top polarized microscopy systems but at much lower cost and complexity. The detection of malaria in fixed and stained blood smears is presented using both, a conventional polarized microscope and our cell-phone based system. The cell-phone based polarimetric microscopy design shows the potential to have both the resolution and specificity to detect malaria in a low-cost, easy-to-use, modular platform.

Malaria Diagnosis Using a Mobile Phone Polarized Microscope

PubMed Central

Pirnstill, Casey W.; Coté, Gerard L.

2015-01-01

Malaria remains a major global health burden, and new methods for low-cost, high-sensitivity, diagnosis are essential, particularly in remote areas with low-resource around the world. In this paper, a cost effective, optical cell-phone based transmission polarized light microscope system is presented for imaging the malaria pigment known as hemozoin. It can be difficult to determine the presence of the pigment from background and other artifacts, even for skilled microscopy technicians. The pigment is much easier to observe using polarized light microscopy. However, implementation of polarized light microscopy lacks widespread adoption because the existing commercial devices have complicated designs, require sophisticated maintenance, tend to be bulky, can be expensive, and would require re-training for existing microscopy technicians. To this end, a high fidelity and high optical resolution cell-phone based polarized light microscopy system is presented which is comparable to larger bench-top polarized microscopy systems but at much lower cost and complexity. The detection of malaria in fixed and stained blood smears is presented using both, a conventional polarized microscope and our cell-phone based system. The cell-phone based polarimetric microscopy design shows the potential to have both the resolution and specificity to detect malaria in a low-cost, easy-to-use, modular platform. PMID:26303238

Less-Costly Ion Implantation of Solar Cells

NASA Technical Reports Server (NTRS)

Fitzgerald, D. J.

1984-01-01

Experiments point way toward more relaxed controls over ion-implanation dosage and uniformity in solar-cell fabrication. Data indicate cell performance, measured by output current density at fixed voltage, virtually same whether implant is particular ion species or broad-beam mixture of several species.

Characterization of atomic spin polarization lifetime of cesium vapor cells with neon buffer gas

NASA Astrophysics Data System (ADS)

Lou, Janet W.; Cranch, Geoffrey A.

2018-02-01

The dephasing time of spin-polarized atoms in an atomic vapor cell plays an important role in determining the stability of vapor-cell clocks as well as the sensitivity of optically-pumped magnetometers. The presence of a buffer gas can extend the lifetime of these atoms. Many vapor cell systems operate at a fixed (often elevated) temperature. For ambient temperature operation with no temperature control, it is necessary to characterize the temperature dependence as well. We present a spin-polarization lifetime study of Cesium vapor cells with different buffer gas pressures, and find good agreement with expectations based on the combined effects of wall collisions, spin exchange, and spin destruction. For our (7.5 mm diameter) vapor cells, the lifetime can be increased by two orders of magnitude by introducing Ne buffer gas up to 100 Torr. Additionally, the dependence of the lifetime on temperature is measured (25 - 47 oC) and simulated for the first time to our knowledge with reasonable agreement.

A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

PubMed

Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W

2014-01-02

Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Copyright © 2014 John Wiley & Sons, Inc.

Influence of Impurities and Filling Protocol on the Aluminum Fixed Point

NASA Astrophysics Data System (ADS)

Renaot, E.; Valin, M. H.; Elgourdou, M.

2008-06-01

To improve the uncertainty of the aluminum fixed point, a study was launched by LNE-INM/CNAM in the framework of the EUROMET Project 732 “Toward more accurate temperature fixed points” (Coordinating laboratory: LNE-INM/CNAM, 17 partner countries). A new open cell was filled with aluminum of 99.99995% purity. A French laboratory carried out elemental analysis of the sample using glow discharge-mass spectrometry (GD-MS). The values of the equilibrium distribution coefficient k and of the derivative {δ T_{{l}}/δ ci_{{l}}} of the temperature of the liquidus line with respect to the concentration of impurity i will be obtained through collaboration with a French physical and chemical laboratory. In the past, some aluminum cells were opened after several melts and freezes. The aluminum ingot was sticking to the graphite crucible, indicating that physicochemical reactions had likely occurred between Al and C. To avoid this reaction, an effort was made to draw benefit from the Al2O3 film that appears immediately on the surface of the aluminum ingot when it is exposed to oxygen. The open aluminum cell was tested in different furnaces and with different thermal insulator arrangements inside the fixed-point assembly. The observed drifts of the plateaux were always larger than the expected values. The cell was opened to inspect the aluminum ingot. The ingot was extracted easily, since no sticking to the crucible had occurred. The aluminum showed a very bright surface, but the presence of many “craters” throughout the thickness of the ingot was surprising. In some cases, the thermometer well was even apparent.

Engineering of a synthetic quadrastable gene network to approach Waddington landscape and cell fate determination

PubMed Central

Wu, Fuqing; Su, Ri-Qi; Lai, Ying-Cheng; Wang, Xiao

2017-01-01

The process of cell fate determination has been depicted intuitively as cells travelling and resting on a rugged landscape, which has been probed by various theoretical studies. However, few studies have experimentally demonstrated how underlying gene regulatory networks shape the landscape and hence orchestrate cellular decision-making in the presence of both signal and noise. Here we tested different topologies and verified a synthetic gene circuit with mutual inhibition and auto-activations to be quadrastable, which enables direct study of quadruple cell fate determination on an engineered landscape. We show that cells indeed gravitate towards local minima and signal inductions dictate cell fates through modulating the shape of the multistable landscape. Experiments, guided by model predictions, reveal that sequential inductions generate distinct cell fates by changing landscape in sequence and hence navigating cells to different final states. This work provides a synthetic biology framework to approach cell fate determination and suggests a landscape-based explanation of fixed induction sequences for targeted differentiation. DOI: http://dx.doi.org/10.7554/eLife.23702.001 PMID:28397688

Microbial Reverse-Electrodialysis Electrolysis and Chemical-Production Cell for H2 Production and CO2 Sequestration.

PubMed

Zhu, Xiuping; Hatzell, Marta C; Logan, Bruce E

2014-04-08

Natural mineral carbonation can be accelerated using acid and alkali solutions to enhance atmospheric CO 2 sequestration, but the production of these solutions needs to be carbon-neutral. A microbial reverse-electrodialysis electrolysis and chemical-production cell (MRECC) was developed to produce these solutions and H 2 gas using only renewable energy sources (organic matter and salinity gradient). Using acetate (0.82 g/L) as a fuel for microorganisms to generate electricity in the anode chamber (liquid volume of 28 mL), 0.45 mmol of acid and 1.09 mmol of alkali were produced at production efficiencies of 35% and 86%, respectively, along with 10 mL of H 2 gas. Serpentine dissolution was enhanced 17-87-fold using the acid solution, with approximately 9 mL of CO 2 absorbed and 4 mg of CO 2 fixed as magnesium or calcium carbonates. The operational costs, based on mineral digging and grinding, and water pumping, were estimated to be only $25/metric ton of CO 2 fixed as insoluble carbonates. Considering the additional economic benefits of H 2 generation and possible wastewater treatment, this method may be a cost-effective and environmentally friendly method for CO 2 sequestration.

Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma.

PubMed

Pettus, Jason R; Wilson, Terri L; Steinmetz, Heather B; Lefferts, Joel A; Tafe, Laura J

2017-02-01

Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting. Copyright © 2016 Elsevier Inc. All rights reserved.

FINE STRUCTURAL LOCALIZATION OF ACYLTRANSFERASES

PubMed Central

Higgins, Joan A.; Barrnett, Russell J.

1971-01-01

A study of the fine structural localization of the acyltransferases of the monoglyceride and α-glycerophosphate pathways for triglyceride synthesis in the intestinal absorptive cell is reported. Glutaraldehyde-fixed tissue was found to synthesize diglyceride and triglyceride from monopalmitin and palmityl CoA, and parallel morphological studies showed the appearance of lipid droplets in the smooth endoplasmic reticulum of the absorptive cell. Glutaraldehyde-fixed tissue also synthesized triglyceride from α-glycerophosphate, although this enzyme system was more susceptible to fixation than the monoglyceride pathway acyltransferases. Cytochemical methods for the localization of free CoA were based (a) on the formation of the insoluble lanthanium mercaptide of CoA and (b) on the reduction of ferricyanide by CoA to yield ferrocyanide which forms an insoluble precipitate with manganous ions. By these methods the monoglyceride pathway acyltransferases were found to be located mainly on the inner surface of the smooth endoplasmic reticulum. The α-glycerophosphate pathway acyltransferases were localized mainly on the rough endoplasmic reticulum. Activity limited to the outer cisternae of the Golgi membranes occurred with both pathways. The possible organization of triglyceride absorption and chylomicron synthesis is discussed in view of these results. PMID:5563442

The Melting Point of Palladium Using Miniature Fixed Points of Different Ceramic Materials: Part II—Analysis of Melting Curves and Long-Term Investigation

NASA Astrophysics Data System (ADS)

Edler, F.; Huang, K.

2016-12-01

Fifteen miniature fixed-point cells made of three different ceramic crucible materials (Al2O3, ZrO2, and Al2O3(86 %)+ZrO2(14 %)) were filled with pure palladium and used to calibrate type B thermocouples (Pt30 %Rh/Pt6 %Rh). A critical point by using miniature fixed points with small amounts of fixed-point material is the analysis of the melting curves, which are characterized by significant slopes during the melting process compared to flat melting plateaus obtainable using conventional fixed-point cells. The method of the extrapolated starting point temperature using straight line approximation of the melting plateau was applied to analyze the melting curves. This method allowed an unambiguous determination of an electromotive force (emf) assignable as melting temperature. The strict consideration of two constraints resulted in a unique, repeatable and objective method to determine the emf at the melting temperature within an uncertainty of about 0.1 μ V. The lifetime and long-term stability of the miniature fixed points was investigated by performing more than 100 melt/freeze cycles for each crucible of the different ceramic materials. No failure of the crucibles occurred indicating an excellent mechanical stability of the investigated miniature cells. The consequent limitation of heating rates to values below {± }3.5 K min^{-1} above 1100° C and the carefully and completely filled crucibles (the liquid palladium occupies the whole volume of the crucible) are the reasons for successfully preventing the crucibles from breaking. The thermal stability of the melting temperature of palladium was excellent when using the crucibles made of Al2O3(86 %)+ZrO2(14 %) and ZrO2. Emf drifts over the total duration of the long-term investigation were below a temperature equivalent of about 0.1 K-0.2 K.

Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative.

PubMed

Duval, Kristin; Aubin, Rémy A; Elliott, James; Gorn-Hondermann, Ivan; Birnboim, H Chaim; Jonker, Derek; Fourney, Ron M; Frégeau, Chantal J

2010-02-01

Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.

Novel Metabolic Attributes of the Genus Cyanothece, Comprising a Group of Unicellular Nitrogen-Fixing Cyanobacteria

PubMed Central

Bandyopadhyay, Anindita; Elvitigala, Thanura; Welsh, Eric; Stöckel, Jana; Liberton, Michelle; Min, Hongtao; Sherman, Louis A.; Pakrasi, Himadri B.

2011-01-01

ABSTRACT The genus Cyanothece comprises unicellular cyanobacteria that are morphologically diverse and ecologically versatile. Studies over the last decade have established members of this genus to be important components of the marine ecosystem, contributing significantly to the nitrogen and carbon cycle. System-level studies of Cyanothece sp. ATCC 51142, a prototypic member of this group, revealed many interesting metabolic attributes. To identify the metabolic traits that define this class of cyanobacteria, five additional Cyanothece strains were sequenced to completion. The presence of a large, contiguous nitrogenase gene cluster and the ability to carry out aerobic nitrogen fixation distinguish Cyanothece as a genus of unicellular, aerobic nitrogen-fixing cyanobacteria. Cyanothece cells can create an anoxic intracellular environment at night, allowing oxygen-sensitive processes to take place in these oxygenic organisms. Large carbohydrate reserves accumulate in the cells during the day, ensuring sufficient energy for the processes that require the anoxic phase of the cells. Our study indicates that this genus maintains a plastic genome, incorporating new metabolic capabilities while simultaneously retaining archaic metabolic traits, a unique combination which provides the flexibility to adapt to various ecological and environmental conditions. Rearrangement of the nitrogenase cluster in Cyanothece sp. strain 7425 and the concomitant loss of its aerobic nitrogen-fixing ability suggest that a similar mechanism might have been at play in cyanobacterial strains that eventually lost their nitrogen-fixing ability. PMID:21972240

47 CFR 90.213 - Frequency stability.

Code of Federal Regulations, 2010 CFR

2010-10-01

... range (MHz) Fixed and base stations Mobile stations Over 2 watts output power 2 watts or less output...-930 1.5 935-940 0.1 1.5 1.5 1427-1435 9 300 300 300 Above 2450 10 1 Fixed and base stations with over... stability of 5 ppm. 5 In the 150-174 MHz band, fixed and base stations with a 12.5 kHz channel bandwidth...

102. DETAIL OF LEAD ROUGHER CELL INTERIOR. NOTE SEVERAL CELLS ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

102. DETAIL OF LEAD ROUGHER CELL INTERIOR. NOTE SEVERAL CELLS COMBINED INTO ONE LARGER CELL FOR THIS STAGE, DIPPER FOR SAMPLING SLIME, AND TWO DIFFERENT STYLES OF GALIGHER "AGITAIR" FIXED BAFFLES AROUND AGITATOR. GALIGHER AGITATORS HAVE PADDLE BLADES ON BOTTOM OF DISKS. - Shenandoah-Dives Mill, 135 County Road 2, Silverton, San Juan County, CO

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

PubMed

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules.

PubMed

Kwiatkowska, Maria; Stępiński, Dariusz; Polit, Justyna T; Popłońska, Katarzyna; Wojtczak, Agnieszka

2011-01-01

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of β-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.

Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining

NASA Astrophysics Data System (ADS)

Adams, Daniel L.; Alpaugh, R. Katherine; Tsai, Susan; Tang, Cha-Mei; Stefansson, Steingrimur

2016-09-01

In tissue biopsies formalin fixed paraffin embedded cancer blocks are micro-sectioned producing multiple semi-identical specimens which are analyzed and subtyped proteomically, and genomically, with numerous biomarkers. In blood based biopsies (BBBs), blood is purified for circulating tumor cells (CTCs) and clinical utility is typically limited to cell enumeration, as only 2-3 positive fluorescent markers and 1 negative marker can be used. As such, increasing the number of subtyping biomarkers on each individual CTC could dramatically enhance the clinical utility of BBBs, allowing in depth interrogation of clinically relevant CTCs. We describe a simple and inexpensive method for quenching the specific fluors of fluorescently stained CTCs followed by sequential restaining with additional biomarkers. As proof of principle a CTC panel, immunosuppression panel and stem cell panel were used to sequentially subtype individual fluorescently stained patient CTCs, suggesting a simple and universal technique to analyze multiple clinically applicable immunomarkers from BBBs.

Hybridoma cell agglutination as a novel test to detect circulating antigen of Schistosoma japonicum.

PubMed

Li, Yong-Long; Liu, Wenqi; Ruppel, Andreas

2003-01-01

We developed a serodiagnostic test which is based on the agglutination of hybridoma cells. In the presence of specific antigen, agglutination of the fixed and stained cells occurs and can be visualized in analogy to traditional erythrocyte agglutination. The procedures were developed with a murine cell line producing a monoclonal antibody against a schistosome gut protein and sera of patients and mice infected with Schistosoma japonicum. This test is capable of detecting circulating antigen during pre-patency in mice infected with 50 cercariae. Its sensitivity was high with acute schistosomiasis japonica (97%, n = 32) and moderate with chronic cases (75%, n = 57). No positive reactions were obtained with healthy persons (n = 78) or patients infected with other parasites (Chlonorchis sinensis, n = 20; Paragonimus westermani, n = 20; Plasmodium vivax, n = 10) or suffering from lupus erythomatodus (n = 5) or mononucleosis (n = 10).

Chemical imaging of a Symbiodinium sp. cell using synchrotron infrared microspectroscopy: a feasibility study.

PubMed

Gordon, B R; Martin, D E; Bambery, K R; Motti, C A

2018-04-01

The symbiotic relationship between corals and Symbiodinium spp. is the key to the success and survival of coral reef ecosystems the world over. Nutrient exchange and chemical communication between the two partners provides the foundation of this key relationship, yet we are far from a complete understanding of these processes. This is due, in part, to the difficulties associated with studying an intracellular symbiosis at the small spatial scales required to elucidate metabolic interactions between the two partners. This feasibility study, which accompanied a more extensive investigation of fixed Symbiodinium cells (data unpublished), examines the potential of using synchrotron radiation infrared microspectroscopy (SR-IRM) for exploring metabolite localisation within a single Symbiodinium cell. In doing so, three chemically distinct subcellular regions of a single Symbiodinium cell were established and correlated to cellular function based on assignment of diagnostic chemical classes. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Azimuthal phase retardation microscope for visualizing actin filaments of biological cells

NASA Astrophysics Data System (ADS)

Shin, In Hee; Shin, Sang-Mo

2011-09-01

We developed a new theory-based azimuthal phase retardation microscope to visualize distributions of actin filaments in biological cells without having them with exogenous dyes, fluorescence labels, or stains. The azimuthal phase retardation microscope visualizes distributions of actin filaments by measuring the intensity variations of each pixel of a charge coupled device camera while rotating a single linear polarizer. Azimuthal phase retardation δ between two fixed principal axes was obtained by calculating the rotation angles of the polarizer at the intensity minima from the acquired intensity data. We have acquired azimuthal phase retardation distributions of human breast cancer cell, MDA MB 231 by our microscope and compared the azimuthal phase retardation distributions with the fluorescence image of actin filaments by the commercial fluorescence microscope. Also, we have observed movement of human umbilical cord blood derived mesenchymal stem cells by measuring azimuthal phase retardation distributions.

The dynamic landscape of the cell nucleus.

PubMed

Austin, Christopher M; Bellini, Michel

2010-01-01

While the cell nucleus was described for the first time almost two centuries ago, our modern view of the nuclear architecture is primarily based on studies from the last two decades. This surprising late start coincides with the development of new, powerful strategies to probe for the spatial organization of nuclear activities in both fixed and live cells. As a result, three major principles have emerged: first, the nucleus is not just a bag filled with nucleic acids and proteins. Rather, many distinct functional domains, including the chromosomes, resides within the confines of the nuclear envelope. Second, all these nuclear domains are highly dynamic, with molecules exchanging rapidly between them and the surrounding nucleoplasm. Finally, the motion of molecules within the nucleoplasm appears to be mostly driven by random diffusion. Here, the emerging roles of several subnuclear domains are discussed in the context of the dynamic functions of the cell nucleus.

Comparison of human driver dynamics in simulators with complex and simple visual displays and in an automobile on the road

NASA Technical Reports Server (NTRS)

Mcruer, D. T.; Klein, R. H.

1975-01-01

As part of a comprehensive program exploring driver/vehicle system response in lateral steering tasks, driver/vehicle system describing functions and other dynamic data have been gathered in several milieu. These include a simple fixed base simulator with an elementary roadway delineation only display; a fixed base statically operating automobile with a terrain model based, wide angle projection system display; and a full scale moving base automobile operating on the road. Dynamic data with the two fixed base simulators compared favorably, implying that the impoverished visual scene, lack of engine noise, and simplified steering wheel feel characteristics in the simple simulator did not induce significant driver dynamic behavior variations. The fixed base vs. moving base comparisons showed substantially greater crossover frequencies and phase margins on the road course.

Quantification of plant cell coupling with live-cell microscopy.

PubMed

Liesche, Johannes; Schulz, Alexander

2015-01-01

Movement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule's capacity to pass a specific cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely determining the plasmodesmata-mediated cell wall permeability for small molecules in living cells.The method is based on photoactivation of the fluorescent tracer caged fluorescein. Non-fluorescent caged fluorescein is applied to a target tissue, where it is taken up passively into all cells. Imaged by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection of three-dimensional (3D) time series. These contain all necessary functional and anatomical data to measure cell coupling in complex tissues noninvasively.

Development of a multiplexed bypass control system for aerospace batteries

NASA Technical Reports Server (NTRS)

Frank, H. A.

1977-01-01

A breadboard bypass control system was developed to control a battery comprised of 26 JPL-developed negative limited Ni-Cd cells. The system was designed to automatically remove cells from the circuit when their voltages exceeded a fixed limit on charge and fell below a fixed limit on discharge. Major components of the system consisted of a cell voltage monitor, a multiplexing circuit, and individual electromechanical relays for each cell. The system was found to function well in controlling the battery during a simulated 10-month MM-71 mission and a 2-month simulated low earth orbit cycling mission. A flight version of the bypass system was estimated to have a total parts count of 150 and total weight of 1.63 kg. When fully developed, the system shows promise for improving life and reliability of spacecraft batteries.

Molecular characterization of oral squamous cell carcinoma using targeted next-generation sequencing.

PubMed

Er, Tze-Kiong; Wang, Yen-Yun; Chen, Chih-Chieh; Herreros-Villanueva, Marta; Liu, Ta-Chih; Yuan, Shyng-Shiou F

2015-10-01

Many genetic factors play an important role in the development of oral squamous cell carcinoma. The aim of this study was to assess the mutational profile in oral squamous cell carcinoma using formalin-fixed, paraffin-embedded tumors from a Taiwanese population by performing targeted sequencing of 26 cancer-associated genes that are frequently mutated in solid tumors. Next-generation sequencing was performed in 50 formalin-fixed, paraffin-embedded tumor specimens obtained from patients with oral squamous cell carcinoma. Genetic alterations in the 26 cancer-associated genes were detected using a deep sequencing (>1000X) approach. TP53, PIK3CA, MET, APC, CDH1, and FBXW7 were most frequently mutated genes. Most remarkably, TP53 mutations and PIK3CA mutations, which accounted for 68% and 18% of tumors, respectively, were more prevalent in a Taiwanese population. Other genes including MET (4%), APC (4%), CDH1 (2%), and FBXW7 (2%) were identified in our population. In summary, our study shows the feasibility of performing targeted sequencing using formalin-fixed, paraffin-embedded samples. Additionally, this study also reports the mutational landscape of oral squamous cell carcinoma in the Taiwanese population. We believe that this study will shed new light on fundamental aspects in understanding the molecular pathogenesis of oral squamous cell carcinoma and may aid in the development of new targeted therapies. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cell-of-Origin in Diffuse Large B-Cell Lymphoma: Are the Assays Ready for the Clinic?

PubMed

Scott, David W

2015-01-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide and consists of a heterogeneous group of cancers classified together on the basis of shared morphology, immunophenotype, and aggressive clinical behavior. It is now recognized that this malignancy comprises at least two distinct molecular subtypes identified by gene expression profiling: the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) groups-the cell-of-origin (COO) classification. These two groups have different genetic mutation landscapes, pathobiology, and outcomes following treatment. Evidence is accumulating that novel agents have selective activity in one or the other COO group, making COO a predictive biomarker. Thus, there is now a pressing need for accurate and robust methods to assign COO, to support clinical trials, and ultimately guide treatment decisions for patients. The "gold standard" methods for COO are based on gene expression profiling (GEP) of RNA from fresh frozen tissue using microarray technology, which is an impractical solution when formalin-fixed paraffin-embedded tissue (FFPET) biopsies are the standard diagnostic material. This review outlines the history of the COO classification before examining the practical implementation of COO assays applicable to FFPET biopsies. The immunohistochemistry (IHC)-based algorithms and gene expression-based assays suitable for the highly degraded RNA from FFPET are discussed. Finally, the technical and practical challenges that still need to be addressed are outlined before robust gene expression-based assays are used in the routine management of patients with DLBCL.

Cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study.

PubMed

Hafez, Hend Salah; Selim, Essam Mohamed Nassef; Kamel Eid, Faten Hussein; Tawfik, Wael Attia; Al-Ashkar, Emad A; Mostafa, Yehya Ahmed

2011-09-01

Treatment with fixed orthodontic appliances in the corrosive environment of the oral cavity warrants in-vivo investigations of biocompatibility. Eighteen control and 28 treated subjects were evaluated longitudinally. Four combinations of brackets and archwires were tested. Buccal mucosa cell samples were collected before treatment, and 3 and 6 months after appliance placement. The cells were processed for cytotoxicity, genotoxicity, and nickel and chromium contents. In the treatment group, buccal mucosa cell viability values were 8.1% at pretreatment, and 6.4% and 4.5% at 3 and 6 months, respectively. The composite score, a calculated DNA damage value, decreased from 125.6 to 98.8 at 6 months. Nickel cellular content increased from 0.52 to 0.68 and 0.78 ng per milliliter, and chromium increased from 0.31 to 0.41 and 0.78 ng per milliliter at 3 and 6 months, respectively. Compared with the control group, the treated subjects showed significant differences for DNA damage and chromium content at 3 months only. Fixed orthodontic appliances decreased cellular viability, induced DNA damage, and increased the nickel and chromium contents of the buccal mucosa cells. Compared to the control group, these changes were not evident at 6 months, possibly indicating tolerance for or repair of the cells and the DNA. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Bilateral Comparison of Mercury and Gallium Fixed-Point Cells Using Standard Platinum Resistance Thermometer

NASA Astrophysics Data System (ADS)

Bojkovski, J.; Veliki, T.; Zvizdić, D.; Drnovšek, J.

2011-08-01

The objective of project EURAMET 1127 (Bilateral comparison of triple point of mercury and melting point of gallium) in the field of thermometry is to compare realization of a triple point of mercury (-38.8344 °C) and melting point of gallium (29.7646 °C) between the Slovenian national laboratory MIRS/UL-FE/LMK and the Croatian national laboratory HMI/FSB-LPM using a long-stem 25 Ω standard platinum resistance thermometer (SPRT). MIRS/UL/FE-LMK participated in a number of intercomparisons on the level of EURAMET. On the other hand, the HMI/LPM-FSB laboratory recently acquired new fixed-point cells which had to be evaluated in the process of intercomparisons. A quartz-sheathed SPRT has been selected and calibrated at HMI/LPM-FSB at the triple point of mercury, the melting point of gallium, and the water triple point. A second set of measurements was made at MIRS/UL/FE-LMK. After its return, the SPRT was again recalibrated at HMI/LPM-FSB. In the comparison, the W value of the SPRT has been used. Results of the bilateral intercomparison confirmed that the new gallium cell of the HMI/LPM-FSB has a value that is within uncertainty limits of both laboratories that participated in the exercise, while the mercury cell experienced problems. After further research, a small leakage in the mercury fixed-point cell has been found.

Optoelectrical modeling of solar cells based on c-Si/a-Si:H nanowire array: focus on the electrical transport in between the nanowires

NASA Astrophysics Data System (ADS)

Levtchenko, Alexandra; Le Gall, Sylvain; Lachaume, Raphaël; Michallon, Jérôme; Collin, Stéphane; Alvarez, José; Djebbour, Zakaria; Kleider, Jean-Paul

2018-06-01

By coupling optical and electrical modeling, we have investigated the photovoltaic performances of p-i-n radial nanowires array based on crystalline p-type silicon (c-Si) core/hydrogenated amorphous silicon (a-Si:H) shell. By varying either the doping concentration of the c-Si core, or back contact work function we can separate and highlight the contribution to the cell’s performance of the nanowires themselves (the radial cell) from the interspace between the nanowires (the planar cell). We show that the build-in potential (V bi) in the radial and planar cells strongly depends on the doping of c-Si core and the work function of the back contact respectively. Consequently, the solar cell’s performance is degraded if either the doping concentration of the c-Si core, or/and the work function of the back contact is too low. By inserting a thin (p) a-Si:H layer between both core/absorber and back contact/absorber, the performance of the solar cell can be improved by partly fixing the V bi at both interfaces due to strong electrostatic screening effect. Depositing such a buffer layer playing the role of an electrostatic screen for charge carriers is a suggested way of enhancing the performance of solar cells based on radial p-i-n or n-i-p nanowire array.

Production of Recombinant Rabies Virus Glycoprotein by Insect Cells in a Single-Use Fixed-Bed Bioreactor.

PubMed

Ventini-Monteiro, Daniella C; Astray, Renato M; Pereira, Carlos A

2018-01-01

A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized. By using the described protocol of production, the following performance can be regularly achieved: 1.7 ± 0.6 × 1E10 total cells; 2.4 ± 0.8 × 1E7 cells/mL and 1.2 ± 0.9 μg of rRVGP/1E7 cells; 1.5 ± 0.8 mg of total rRVGP.

Establishing contact between cell-laden hydrogels and metallic implants with a biomimetic adhesive for cell therapy supported implants.

PubMed

Barthes, Julien; Mutschler, Angela; Dollinger, Camille; Gaudinat, Guillaume; Lavalle, Philippe; Le Houerou, Vincent; Brian McGuinness, Garrett; Engin Vrana, Nihal

2017-12-15

For in-dwelling implants, controlling the biological interface is a crucial parameter to promote tissue integration and prevent implant failure. For this purpose, one possibility is to facilitate the establishment of the interface with cell-laden hydrogels fixed to the implant. However, for proper functioning, the stability of the hydrogel on the implant should be ensured. Modification of implant surfaces with an adhesive represents a promising strategy to promote the adhesion of a cell-laden hydrogel on an implant. Herein, we developed a peptidic adhesive based on mussel foot protein (L-DOPA-L-lysine) 2 -L-DOPA that can be applied directly on the surface of an implant. At physiological pH, unoxidized (L-DOPA-L-lysine) 2 -L-DOPA was supposed to strongly adhere to metallic surfaces but it only formed a very thin coating (less than 1 nm). Once oxidized at physiological pH, (L-DOPA-L-lysine) 2 -L-DOPA forms an adhesive coating about 20 nm thick. In oxidized conditions, L-lysine can adhere to metallic substrates via electrostatic interaction. Oxidized L-DOPA allows the formation of a coating through self-polymerization and can react with amines so that this adhesive can be used to fix extra-cellular matrix based materials on implant surfaces through the reaction of quinones with amino groups. Hence, a stable interface between a soft gelatin hydrogel and metallic surfaces was achieved and the strength of adhesion was investigated. We have shown that the adhesive is non-cytotoxic to encapsulated cells and enabled the adhesion of gelatin soft hydrogels for 21 days on metallic substrates in liquid conditions. The adhesion properties of this anchoring peptide was quantified by a 180° peeling test with a more than 60% increase in peel strength in the presence of the adhesive. We demonstrated that by using a biomimetic adhesive, for the application of cell-laden hydrogels to metallic implant surfaces, the hydrogel/implant interface can be ensured without relying on the properties of the deposited biomaterials.

Computer simulation of a cellular automata model for the immune response in a retrovirus system

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

Pandey, R.B.

1989-02-01

Immune response in a retrovirus system is modeled by a network of three binary cell elements to take into account some of the main functional features of T4 cells, T8 cells, and viruses. Two different intercell interactions are introduced, one of which leads to three fixed points while the other yields bistable fixed points oscillating between a healthy state and a sick state in a mean field treatment. Evolution of these cells is studied for quenched and annealed random interactions on a simple cubic lattice with a nearest neighbor interaction using inhomogenous cellular automata. Populations of T4 cells and viralmore » cells oscillate together with damping (with constant amplitude) for annealed (quenched) interaction on increasing the value of mixing probability B from zero to a characteristic value B/sub ca/ (B/sub cq/). For higher B, the average number of T4 cells increases while that of the viral infected cells decreases monotonically on increasing B, suggesting a phase transition at B/sub ca/ (B/sub cq/).« less

Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.

PubMed

Yamamoto, Tetsushi; Kudo, Mitsuhiro; Peng, Wei-Xia; Takata, Hideyuki; Takakura, Hideki; Teduka, Kiyoshi; Fujii, Takenori; Mitamura, Kuniko; Taga, Atsushi; Uchida, Eiji; Naito, Zenya

2016-10-01

Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain

PubMed Central

Lang, Claus; Smith, Lucinda S.; Haney, Cara H.; Long, Sharon R.

2018-01-01

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a PexoY-mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a PbacA-mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a PnifH-uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context. PMID:29467773

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

PubMed

Lang, Claus; Smith, Lucinda S; Long, Sharon R

2018-01-01

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a P exoY -mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a P bacA -mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a P nifH -uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context.

Pre-labeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

PubMed

Bjerneld, Erik J; Johansson, Johan D; Laurin, Ylva; Hagner-McWhirter, Åsa; Rönn, Ola; Karlsson, Robert

2015-09-01

A pre-labeling protocol based on Cy5 N-hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range-more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250pmol in 20μl using a Tris-HCl labeling buffer at pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of sub-nanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS-PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. Two-dimensional electrophoresis was used to examine the labeling of proteins in a cell lysate using both sulfonated and non-sulfonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points. Copyright © 2015 Elsevier Inc. All rights reserved.

Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs

PubMed Central

Whitford, Margaret H.; Arruda, Valder R.; Stedman, Hansell H.; Kay, Mark A.; High, Katherine A.

2015-01-01

Abstract Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

PubMed

Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

2015-03-01

Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

PubMed

Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

2015-03-15

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. Published by Elsevier Inc.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

USGS Publications Warehouse

Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

2015-01-01

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

Bio-based products from solar energy and carbon dioxide.

PubMed

Yu, Jian

2014-01-01

Producing bio-based products directly from CO₂ and solar energy is a desirable alternative to the conventional biorefining that relies on biomass feedstocks. The production paradigm is based on an artificial photosynthetic system that converts sunlight to electricity and H₂ via water electrolysis. An autotrophic H₂-oxidizing bacterium fixes CO₂ in dark conditions. The assimilated CO₂ is stored in bacterial cells as polyhydroxybutyrate (PHB), from which a range of products can be derived. Compared with natural photosynthesis of a fast-growing cyanobacterium, the artificial photosynthetic system has much higher energy efficiency and productivity of bio-based products. The new technology looks promising because of possible cost reduction in feedstock, equipment, and operation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cell-based quantification of biomarkers from an ultra-fast microfluidic immunofluorescent staining: application to human breast cancer cell lines

NASA Astrophysics Data System (ADS)

Migliozzi, D.; Nguyen, H. T.; Gijs, M. A. M.

2018-02-01

Immunohistochemistry (IHC) is one of the main techniques currently used in the clinics for biomarker characterization. It consists in colorimetric labeling with specific antibodies followed by microscopy analysis. The results are then used for diagnosis and therapeutic targeting. Well-known drawbacks of such protocols are their limited accuracy and precision, which prevent the clinicians from having quantitative and robust IHC results. With our work, we combined rapid microfluidic immunofluorescent staining with efficient image-based cell segmentation and signal quantification to increase the robustness of both experimental and analytical protocols. The experimental protocol is very simple and based on fast-fluidic-exchange in a microfluidic chamber created on top of the formalin-fixed-paraffin-embedded (FFPE) slide by clamping it a silicon chip with a polydimethyl siloxane (PDMS) sealing ring. The image-processing protocol is based on enhancement and subsequent thresholding of the local contrast of the obtained fluorescence image. As a case study, given that the human epidermal growth factor receptor 2 (HER2) protein is often used as a biomarker for breast cancer, we applied our method to HER2+ and HER2- cell lines. We report very fast (5 minutes) immunofluorescence staining of both HER2 and cytokeratin (a marker used to define the tumor region) on FFPE slides. The image-processing program can segment cells correctly and give a cell-based quantitative immunofluorescent signal. With this method, we found a reproducible well-defined separation for the HER2-to-cytokeratin ratio for positive and negative control samples.

Monoclonal antibody (AFH1) immunoreactive on morphologically abnormal basal melanocytes within dysplastic nevi, nevocellular nevus nests, and melanoma.

PubMed

Aronson, P J; Ito, K; Fukaya, T; Hashimoto, K; Mehregan, A H

1988-04-01

The mouse monoclonal antibody AFH1 was produced using formalin-fixed, sham paraffin-embedded human melanoma cell culture line A375 as immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against formalin- or acid alcohol-fixed paraffin-embedded tissue as well as formalin- or acid alcohol-fixed unembedded lesions. Ninety-seven nevomelanocytic lesions, neurofibromas, epithelial lesions, and a plasmacellular infiltrate were evaluated. AFH1 was immunoreactive on 54 of 55 nevocytic lesions (98.2%), 15 of 16 primary melanomas (93.7%), a lentigo maligna, and nests in 21 of 21 dysplastic nevi (100%). Of 100 consecutive basal melanocytes of intraepidermal melanoma cells counted in each lesion, mean AFH1 immunoreactivity for nonnested basal melanocytes in nevocellular nevi was 3.8%; for dysplastic nevi, 13.8%; and for intraepidermal melanoma cells, 78.0%. When nonnested basal melanocytes were subdivided into cytologically normal and abnormal cell groups, AFH1 immunoreactivity was 9.4% and 72.6%, respectively. AFH1 recognition of the lentiginous portion of dysplastic nevi corresponds statistically to the appearance of abnormal melanocyte cytology, nest formation, or both. Using 50% immunoreactive nonnested melanocytes as the criterion, AFH1 seems to distinguish primary melanoma from dysplastic nevi with a sensitivity of 93.8% and a specificity of 95.8%.

The Symbiosome: Legume and Rhizobia Co-evolution toward a Nitrogen-Fixing Organelle?

PubMed Central

Coba de la Peña, Teodoro; Fedorova, Elena; Pueyo, José J.; Lucas, M. Mercedes

2018-01-01

In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation, these bacteria develop mutual metabolic dependence with the host legume. In most legumes, the rhizobia infect post-mitotic cells that have lost their ability to divide, although in some nodules cells do maintain their mitotic capacity after infection. Here, we review what is currently known about legume symbiosomes from an evolutionary and developmental perspective, and in the context of the different interactions between diazotroph bacteria and eukaryotes. As a result, it can be concluded that the symbiosome possesses organelle-like characteristics due to its metabolic behavior, the composite origin and differentiation of its membrane, the retargeting of host cell proteins, the control of microsymbiont proliferation and differentiation by the host legume, and the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells. Different degrees of symbiosome evolution can be defined, specifically in relation to rhizobial infection and to the different types of nodule. Thus, our current understanding of the symbiosome suggests that it might be considered a nitrogen-fixing link in organelle evolution and that the distinct types of legume symbiosomes could represent different evolutionary stages toward the generation of a nitrogen-fixing organelle. PMID:29403508

Wall shear stress fixed points in cardiovascular fluid mechanics.

PubMed

Arzani, Amirhossein; Shadden, Shawn C

2018-05-17

Complex blood flow in large arteries creates rich wall shear stress (WSS) vectorial features. WSS acts as a link between blood flow dynamics and the biology of various cardiovascular diseases. WSS has been of great interest in a wide range of studies and has been the most popular measure to correlate blood flow to cardiovascular disease. Recent studies have emphasized different vectorial features of WSS. However, fixed points in the WSS vector field have not received much attention. A WSS fixed point is a point on the vessel wall where the WSS vector vanishes. In this article, WSS fixed points are classified and the aspects by which they could influence cardiovascular disease are reviewed. First, the connection between WSS fixed points and the flow topology away from the vessel wall is discussed. Second, the potential role of time-averaged WSS fixed points in biochemical mass transport is demonstrated using the recent concept of Lagrangian WSS structures. Finally, simple measures are proposed to quantify the exposure of the endothelial cells to WSS fixed points. Examples from various arterial flow applications are demonstrated. Copyright © 2018 Elsevier Ltd. All rights reserved.

The response of single human cells to zero-gravity

NASA Technical Reports Server (NTRS)

Montgomery, P. O., Jr.; Cook, J. E.; Reynolds, R. C.; Paul, J. S.; Hayflick, L.; Stock, D.; Shulz, W. W.; Kimzey, S. L.; Thirolf, R. G.; Rogers, T.

1977-01-01

Microscopic and histochemical evaluations of human embrionic lung cells after exposure to zero-gravity are reported. Growth curves, DNA microspectrophotometry, phase microscopy, and ultrastructural studies of fixed cells revealed no effects on the cultures. Minor unexplained differences have been found in biochemical constituents of the samples.

Submersed sensing electrode used in fuel-cell type hydrogen detector

NASA Technical Reports Server (NTRS)

Niedrach, L. W.; Rudek, F. P.; Rutkoneski, M. D.

1971-01-01

Electrode has silicone rubber diffusion barrier with fixed permeation constant for hydrogen. Barrier controls flow of hydrogen to anode and Faraday relationship establishes upper limit for current through cell. Electrode fabrication is described.

ConSpeciFix: Classifying prokaryotic species based on gene flow.

PubMed

Bobay, Louis-Marie; Ellis, Brian Shin-Hua; Ochman, Howard

2018-05-16

Classification of prokaryotic species is usually based on sequence similarity thresholds, which are easy to apply but lack a biologically-relevant foundation. Here, we present ConSpeciFix, a program that classifies prokaryotes into species using criteria set forth by the Biological Species Concept, thereby unifying species definition in all domains of life. ConSpeciFix's webserver is freely available at www.conspecifix.com. The local version of the program can be freely downloaded from https://github.com/Bobay-Ochman/ConSpeciFix. ConSpeciFix is written in Python 2.7 and requires the following dependencies: Usearch, MCL, MAFFT and RAxML. ljbobay@uncg.edu.

Fixed-time stabilization of impulsive Cohen-Grossberg BAM neural networks.

PubMed

Li, Hongfei; Li, Chuandong; Huang, Tingwen; Zhang, Wanli

2018-02-01

This article is concerned with the fixed-time stabilization for impulsive Cohen-Grossberg BAM neural networks via two different controllers. By using a novel constructive approach based on some comparison techniques for differential inequalities, an improvement theorem of fixed-time stability for impulsive dynamical systems is established. In addition, based on the fixed-time stability theorem of impulsive dynamical systems, two different control protocols are designed to ensure the fixed-time stabilization of impulsive Cohen-Grossberg BAM neural networks, which include and extend the earlier works. Finally, two simulations examples are provided to illustrate the validity of the proposed theoretical results. Copyright © 2017 Elsevier Ltd. All rights reserved.

Experiment K-7-16: Effects of Microgravity or Simulated Launch on Testicular Function in Rats

NASA Technical Reports Server (NTRS)

Amann, R. P.; Clemens, J. W.; Deaver, D.; Folmer, J.; Zirkin, B.; Veeramachaneni, D. N. R.; Grills, G. S.; Gruppi, C. M.; Wolgemuth, D.; Serova, L. V.;

Electrochemically and Bioelectrochemically Induced Ammonium Recovery

PubMed Central

Gildemyn, Sylvia; Luther, Amanda K.; Andersen, Stephen J.; Desloover, Joachim; Rabaey, Korneel

2015-01-01

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ammonium from an ammonium-rich stream is demonstrated using an electrochemical and a bioelectrochemical system. Both systems are controlled by a potentiostat to either fix the current (for the electrochemical cell) or fix the potential of the working electrode (for the bioelectrochemical cell). In the bioelectrochemical cell, electroactive bacteria catalyze the anodic reaction, whereas in the electrochemical cell the potentiostat applies a higher voltage to produce a current. The current and consequent restoration of the charge balance across the cell allow the transport of cations, such as ammonium, across a cation exchange membrane from the anolyte to the catholyte. The high pH of the catholyte leads to formation of ammonia, which can be stripped from the medium and captured in an acid solution, thus enabling the recovery of a valuable nutrient. The flux of ammonium across the membrane is characterized at different anolyte ammonium concentrations and currents for both the abiotic and biotic reactor systems. Both systems are compared based on current and removal efficiencies for ammonium, as well as the energy input required to drive ammonium transfer across the cation exchange membrane. Finally, a comparative analysis considering key aspects such as reliability, electrode cost, and rate is made. This video article and protocol provide the necessary information to conduct electrochemical and bioelectrochemical ammonia recovery experiments. The reactor setup for the two cases is explained, as well as the reactor operation. We elaborate on data analysis for both reactor types and on the advantages and disadvantages of bioelectrochemical and electrochemical systems. PMID:25651406

Electrochemically and bioelectrochemically induced ammonium recovery.

PubMed

Gildemyn, Sylvia; Luther, Amanda K; Andersen, Stephen J; Desloover, Joachim; Rabaey, Korneel

2015-01-22

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ammonium from an ammonium-rich stream is demonstrated using an electrochemical and a bioelectrochemical system. Both systems are controlled by a potentiostat to either fix the current (for the electrochemical cell) or fix the potential of the working electrode (for the bioelectrochemical cell). In the bioelectrochemical cell, electroactive bacteria catalyze the anodic reaction, whereas in the electrochemical cell the potentiostat applies a higher voltage to produce a current. The current and consequent restoration of the charge balance across the cell allow the transport of cations, such as ammonium, across a cation exchange membrane from the anolyte to the catholyte. The high pH of the catholyte leads to formation of ammonia, which can be stripped from the medium and captured in an acid solution, thus enabling the recovery of a valuable nutrient. The flux of ammonium across the membrane is characterized at different anolyte ammonium concentrations and currents for both the abiotic and biotic reactor systems. Both systems are compared based on current and removal efficiencies for ammonium, as well as the energy input required to drive ammonium transfer across the cation exchange membrane. Finally, a comparative analysis considering key aspects such as reliability, electrode cost, and rate is made. This video article and protocol provide the necessary information to conduct electrochemical and bioelectrochemical ammonia recovery experiments. The reactor setup for the two cases is explained, as well as the reactor operation. We elaborate on data analysis for both reactor types and on the advantages and disadvantages of bioelectrochemical and electrochemical systems.

Continuous uniformly finite time exact disturbance observer based control for fixed-time stabilization of nonlinear systems with mismatched disturbances

PubMed Central

Liu, Chongxin; Liu, Hang

2017-01-01

This paper presents a continuous composite control scheme to achieve fixed-time stabilization for nonlinear systems with mismatched disturbances. The composite controller is constructed in two steps: First, uniformly finite time exact disturbance observers are proposed to estimate and compensate the disturbances. Then, based on adding a power integrator technique and fixed-time stability theory, continuous fixed-time stable state feedback controller and Lyapunov functions are constructed to achieve global fixed-time system stabilization. The proposed control method extends the existing fixed-time stable control results to high order nonlinear systems with mismatched disturbances and achieves global fixed-time system stabilization. Besides, the proposed control scheme improves the disturbance rejection performance and achieves performance recovery of nominal system. Simulation results are provided to show the effectiveness, the superiority and the applicability of the proposed control scheme. PMID:28406966

10 CFR 603.305 - Use of a fixed-support TIA.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Use of a fixed-support TIA. 603.305 Section 603.305 Energy... Expenditure-Based and Fixed-Support Technology Investment Agreements § 603.305 Use of a fixed-support TIA. The contracting officer may use a fixed-support TIA if: (a) The agreement is to support or stimulate RD&D with...

32 CFR 37.300 - What is the difference between an expenditure-based and fixed-support TIA?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 1 2011-07-01 2011-07-01 false What is the difference between an expenditure-based and fixed-support TIA? 37.300 Section 37.300 National Defense Department of Defense OFFICE OF THE... and Fixed-Support Technology Investment Agreements § 37.300 What is the difference between an...

Hyperchromatic laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

PubMed Central

Orabona, Emanuele; De Stefano, Luca; Ferry, Mike; Hasty, Jeff; di Bernardo, Mario; di Bernardo, Diego

2014-01-01

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available. PMID:24831205

Mast cell concentration and skin wound contraction in rats treated with Brazilian pepper essential oil (Schinus terebinthifolius Raddi).

PubMed

Estevão, Lígia Reis Moura; Medeiros, Juliana Pinto de; Simões, Ricardo Santos; Arantes, Rosa Maria Esteves; Rachid, Milene Alvarenga; Silva, Regildo Márcio Gonçalves da; Mendonça, Fábio de Souza; Evêncio-Neto, Joaquim

2015-04-01

To evaluate wound contraction and the concentration of mast cells in skin wounds treated with 5% BPT essential oil-based ointment in rats. Twenty rats, male, of adult age, were submitted to skin surgery on the right (RA) and left antimeres (LA) of the thoracic region. They were divided into two groups: control (RA - wounds receiving daily topical application of vaseline and lanolin) and treated (LA - wounds treated daily with the topical ointment). The skin region with wounds were collected at days 4, 7, 14 and 21 after surgery. Those were fixed in 10% formaldehyde and later processed for paraffin embedding. Sections were obtained and stained by H.E for histopathology analysis. The degree of epithelial contraction was measured and mast cell concentration were also evaluated. The treated group showed higher mast cell concentrations (p<0.05) associated with increased contraction at day 7 and 14 respectively. Ointment containing 5% Brazilian pepper tree oil increases mast cell concentration and promotes skin wound contraction in rats.

Formation and maintenance of nitrogen-fixing cell patterns in filamentous cyanobacteria.

PubMed

Muñoz-García, Javier; Ares, Saúl

2016-05-31

Cyanobacteria forming one-dimensional filaments are paradigmatic model organisms of the transition between unicellular and multicellular living forms. Under nitrogen-limiting conditions, in filaments of the genus Anabaena, some cells differentiate into heterocysts, which lose the possibility to divide but are able to fix environmental nitrogen for the colony. These heterocysts form a quasiregular pattern in the filament, representing a prototype of patterning and morphogenesis in prokaryotes. Recent years have seen advances in the identification of the molecular mechanism regulating this pattern. We use these data to build a theory on heterocyst pattern formation, for which both genetic regulation and the effects of cell division and filament growth are key components. The theory is based on the interplay of three generic mechanisms: local autoactivation, early long-range inhibition, and late long-range inhibition. These mechanisms can be identified with the dynamics of hetR, patS, and hetN expression. Our theory reproduces quantitatively the experimental dynamics of pattern formation and maintenance for wild type and mutants. We find that hetN alone is not enough to play the role as the late inhibitory mechanism: a second mechanism, hypothetically the products of nitrogen fixation supplied by heterocysts, must also play a role in late long-range inhibition. The preponderance of even intervals between heterocysts arises naturally as a result of the interplay between the timescales of genetic regulation and cell division. We also find that a purely stochastic initiation of the pattern, without a two-stage process, is enough to reproduce experimental observations.

The multicellular nature of filamentous heterocyst-forming cyanobacteria.

PubMed

Herrero, Antonia; Stavans, Joel; Flores, Enrique

2016-11-01

Cyanobacteria carry out oxygenic photosynthesis, play a key role in the cycling of carbon and nitrogen in the biosphere, and have had a large impact on the evolution of life and the Earth itself. Many cyanobacterial strains exhibit a multicellular lifestyle, growing as filaments that can be hundreds of cells long and endowed with intercellular communication. Furthermore, under depletion of combined nitrogen, filament growth requires the activity of two interdependent cell types: vegetative cells that fix CO2 and heterocysts that fix N2. Intercellular molecular transfer is essential for signaling involved in the regulation of heterocyst differentiation and for reciprocal nutrition of heterocysts and vegetative cells. Here we review various aspects of multicellularity in cyanobacterial filaments and their differentiation, including filament architecture with emphasis on the structures used for intercellular communication; we survey theoretical models that have been put forward to understand heterocyst patterning and discuss the factors that need to be considered for these models to reflect the biological entity; and finally, since cell division in filamentous cyanobacteria has the peculiarity of producing linked instead of independent cells, we review distinct aspects of cell division in these organisms.

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

PubMed

Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

2012-06-05

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

Adherence of carp leucocytes to adults and cercariae of the blood fluke Sanguinicola inermis.

PubMed

Richards, D T; Hoole, D; Lewis, J W; Ewens, E; Arme, C

1996-03-01

Live adult and cercarial stages of Sanguinicola inermis Plehn, 1905 (Trematoda:Sanguinicolidae) were maintained in vitro in the presence of carp (Cyprinus carpio L.) leucocytes. Cells and parasites were fixed at intervals from 0.25 to 48 h and examined using light microscopy, SEM and TEM. Within 12 h of exposure, leucocytes were found attached to cercariae although, by 24 h, fewer cells were found attached to postcercarial, juvenile adult stages that had shed their tails. Neutrophils and macrophages were found attached to the damaged tegument of cercarie that had not transformed by 48 h. Few cells were attached to the tegument of adult flukes that were alive when fixed. However, there was extensive tegumental damage and numerous cells were attached to adult flukes that had died before fixation. The results are discussed with reference to parasite survival within the vascular system of the host.

Bluetooth wireless monitoring, diagnosis and calibration interface for control system of fuel cell bus in Olympic demonstration

NASA Astrophysics Data System (ADS)

Hua, Jianfeng; Lin, Xinfan; Xu, Liangfei; Li, Jianqiu; Ouyang, Minggao

With the worldwide deterioration of the natural environment and the fossil fuel crisis, the possible commercialization of fuel cell vehicles has become a hot topic. In July 2008, Beijing started a clean public transportation plan for the 29th Olympic games. Three fuel cell city buses and 497 other low-emission vehicles are now serving the Olympic core area and Beijing urban areas. The fuel cell buses will operate along a fixed bus line for 1 year as a public demonstration of green energy vehicles. Due to the specialized nature of fuel cell engines and electrified power-train systems, measurement, monitoring and calibration devices are indispensable. Based on the latest Bluetooth wireless technology, a novel Bluetooth universal data interface was developed for the control system of the fuel cell city bus. On this platform, a series of wireless portable control auxiliary systems have been implemented, including wireless calibration, a monitoring system and an in-system programming platform, all of which are ensuring normal operation of the fuel cell buses used in the demonstration.

Characterization and Identification of Phosphate Solubilizing Bacteria Isolate GPC3.7 from Limestone Mining Region

NASA Astrophysics Data System (ADS)

Fitriyanti, D.; Mubarik, N. R.; Tjahjoleksono, A.

2017-03-01

Phosphate (P) are one of major macronutrients needed by plants. P in the soil are present in the organic and inorganic form. The amounts of P in marginal soil can be increased with plant growth promoting rhizobacteria (PGPR). The aim of this study was to characterize and identify P solubilizing bacteria (PSB) isolate GPC3.7 that characteristically could fix N from the soil around limestone mining area. There were 44 PSB isolates found from 15 soil samples around limestone mining area, Blindis mountain, Cirebon. The solubility index of all strain were measured about 0.125 to 2.375 on Pikovskaya media. There were 22 PSB isolates were grown on N-free bromothymol blue (NfB) medium and 19 isolates were grown on Congo Red Agar (CRA) medium. Only 10 isolates were indicated as symbiotic living microorganisms whereas 12 others were categorized as N-free fixing bacteria. Isolate GPC3.7 was chosen to be further observed, based on its P solubility index, N-fixing ability and growth stability. Phosphate quantitative estimation assay of isolate GPC3.7 was unmeasured. The P soluble concentration of GPC3.7 might be lower than 1 mg/L. The colony of GPC3.7 morphologically had round shape, entire margin, raised elevation and white color. Isolate GPC3.7 was Gram negative bacteria with coccus cell shape. Based on 16S rRNA gene, GPC3.7 was closely relative to Acinetobacter baumannii.

On Determining if Tree-based Networks Contain Fixed Trees.

PubMed

Anaya, Maria; Anipchenko-Ulaj, Olga; Ashfaq, Aisha; Chiu, Joyce; Kaiser, Mahedi; Ohsawa, Max Shoji; Owen, Megan; Pavlechko, Ella; St John, Katherine; Suleria, Shivam; Thompson, Keith; Yap, Corrine

2016-05-01

We address an open question of Francis and Steel about phylogenetic networks and trees. They give a polynomial time algorithm to decide if a phylogenetic network, N, is tree-based and pose the problem: given a fixed tree T and network N, is N based on T? We show that it is [Formula: see text]-hard to decide, by reduction from 3-Dimensional Matching (3DM) and further that the problem is fixed-parameter tractable.

High resolution Fourier interferometer-spectrophotopolarimeter

NASA Technical Reports Server (NTRS)

Fymat, A. L. (Inventor)

1976-01-01

A high-resolution Fourier interferometer-spectrophotopolarimeter is provided using a single linear polarizer-analyzer the transmission axis azimuth of which is positioned successively in the three orientations of 0 deg, 45 deg, and 90 deg, in front of a detector; four flat mirrors, three of which are switchable to either of two positions to direct an incoming beam from an interferometer to the polarizer-analyzer around a sample cell transmitted through a medium in a cell and reflected by medium in the cell; and four fixed focussing lenses, all located in a sample chamber attached at the exit side of the interferometer. This arrangement can provide the distribution of energy and complete polarization state across the spectrum of the reference light entering from the interferometer; the same light after a fixed-angle reflection from the sample cell containing a medium to be analyzed; and the same light after direct transmission through the same sample cell, with the spectral resolution provided by the interferometer.

New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

PubMed

Vedyaykin, Alexey D; Vishnyakov, Innokentii E; Polinovskaya, Vasilisa S; Khodorkovskii, Mikhail A; Sabantsev, Anton V

2016-06-01

FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules.

PubMed

Tétart, F; Bouché, J P

1992-03-01

We show that the 53-nucleotide RNA molecule encoded by gene dicF blocks cell division in Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome-binding region of the ftsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ-lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.

Characterization of an Adhesion-Associated Tumor Suppressor in Breast Cancer

DTIC Science & Technology

2001-08-01

Western blot analysis were invasive and associated with fibrous connective tissue (Fig. 4, B of whole cell lysates resolved by SDS-PAGE was...of breast cancer. Immunohistochemical analyses of archival, formalin-fixed paraffin-embedded specimens of benign and malignant breast tissues confirm...10A cells. In particular, EphA2 destabilizes cell-cell attachments while increasing cell interactions with extracellular matrix (ECM proteins). We have

High-Throughput Sequencing and Copy Number Variation Detection Using Formalin Fixed Embedded Tissue in Metastatic Gastric Cancer

PubMed Central

Hong, Min Eui; Do, In-Gu; Kang, So Young; Ha, Sang Yun; Kim, Seung Tae; Park, Se Hoon; Kang, Won Ki; Choi, Min-Gew; Lee, Jun Ho; Sohn, Tae Sung; Bae, Jae Moon; Kim, Sung; Kim, Duk-Hwan; Kim, Kyoung-Mee

2014-01-01

In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%), APC (10.1%), PIK3CA (5.6%), KRAS (4.5%), SMO (3.4%), STK11 (3.4%), CDKN2A (3.4%) and SMAD4 (3.4%). Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%), 4 (4.5%), 2 (2.2%), 1 (1.1%) and 1 (1.1%) cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes. PMID:25372287

47 CFR 90.419 - Points of communication.

Code of Federal Regulations, 2010 CFR

2010-10-01

... communicate between associated mobile stations and associated base stations of the licensee. Accordingly, operations between base stations at fixed locations are permitted only in the following situations: (a) Base... frequencies below 450 MHz, may communicate on a secondary basis with other base stations, operational fixed...

Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

PubMed

Pollex, Tim; Piolot, Tristan; Heard, Edith

2013-01-01

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates

PubMed Central

1977-01-01

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D- galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies. PMID:833547

Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses

PubMed Central

Hauck, Bernd; Murphy, Samuel L; Smith, Peter H; Qu, Guang; Liu, Xingge; Zelenaia, Olga; Mingozzi, Federico; Sommer, Jürg M; High, Katherine A; Wright, J. Fraser

2008-01-01

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid–specific CD8+ T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription–PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual AmpR, and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8+ T cells that eliminated vector-transduced cells in the clinical study. PMID:18941440

Investigation of TiC C Eutectic and WC C Peritectic High-Temperature Fixed Points

NASA Astrophysics Data System (ADS)

Sasajima, Naohiko; Yamada, Yoshiro

2008-06-01

TiC C eutectic (2,761°C) and WC C peritectic (2,749°C) fixed points were investigated to compare their potential as high-temperature thermometric reference points. Two TiC C and three WC C fixed-point cells were constructed, and the melting and freezing plateaux were evaluated by means of radiation thermometry. The repeatability of the TiC C eutectic within a day was 60 mK with a melting range roughly 200 mK. The repeatability of the melting temperature of the WC C peritectic within 1 day was 17 mK with a melting range of ˜70 mK. The repeatability of the freezing temperature of the WC C peritectic was 21 mK with a freezing range less than 20 mK. One of the TiC C cells was constructed from a TiC and graphite powder mixture. The filling showed the reaction with the graphite crucible was suppressed and the ingot contained less voids, although the lack of high-purity TiC powder poses a problem. The WC C cells were easily constructed, like metal carbon eutectic cells, without any evident reaction with the crucible. From these results, it is concluded that the WC C peritectic has more potential than the TiC C eutectic as a high-temperature reference point. The investigation of the purification of the TiC C cell during filling and the plateau observation are also reported.

Real-time detection of BRAF V600E mutation from archival hairy cell leukemia FFPE tissue by nanopore sequencing.

PubMed

Vacca, Davide; Cancila, Valeria; Gulino, Alessandro; Lo Bosco, Giosuè; Belmonte, Beatrice; Di Napoli, Arianna; Florena, Ada Maria; Tripodo, Claudio; Arancio, Walter

2018-02-01

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.

Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

PubMed Central

Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

1992-01-01

Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306

Landsat 8 Data Modeled as DGGS Data Cubes

NASA Astrophysics Data System (ADS)

Sherlock, M. J.; Tripathi, G.; Samavati, F.

2016-12-01

In the context of tracking recent global changes in the Earth's landscape, Landsat 8 provides high-resolution multi-wavelength data with a temporal resolution of sixteen days. Such a live dataset can benefit novel applications in environmental monitoring. However, a temporal analysis of this dataset in its native format is a challenging task mostly due to the huge volume of geospatial images and imperfect overlay of different day Landsat 8 images. We propose the creation of data cubes derived from Landsat 8 data, through the use of a Discrete Global Grid System (DGGS). DGGS referencing of Landsat 8 data provides a cell-based representation of the pixel values for a fixed area on earth, indexed by keys. Having the calibrated cell-based Landsat 8 images can speed up temporal analysis and facilitate parallel processing using distributed systems. In our method, the Landsat 8 dataset hosted on Amazon Web Services (AWS) is downloaded using a web crawler and stored on a filesystem. We apply the cell-based DGGS referencing (using Pyxis SDK) to Landsat 8 images which provide a rhombus based tessellation of equal area cells for our use-case. After this step, the cell-images which overlay perfectly on different days, are stacked in the temporal dimension and stored into data cube units. The depth of the cube represents the number of temporal images of the same cell and can be updated when new images are received each day. Harnessing the regular spatio-temporal structure of data cubes, we want to compress, query, transmit and visualize big Landsat 8 data in an efficient way for temporal analysis.

Auxins upregulate nif and fix genes.

PubMed

Bianco, Carmen; Defez, Roberto

2010-10-01

In a recent publication we analyzed the global effects triggered by IAA overproduction in S. meliloti RD64 under free-living conditions by comparing the gene expression pattern of wild type 1021 with that of RD64 and 1021 treated with IAA and other four chemically or functionally related molecules. Among the genes differentially expressed in RD64 and IAA-treated 1021 cells we found two genes of pho operon, phoT and phoC. Based on this finding we examined the mechanisms for mineral P solubilization in RD64 and the potential ability of this strain to improve Medicago growth under P-starved conditions. Here, we further analyze the expression profiles obtained in microarray analysis and evaluate the specificity and the extent of overlap between all treatments. Venn diagrams indicated that IAA- and 2,4-D-regulated genes were closely related. Furthermore, most differentially expressed genes from pSymA were induced in 1021 cells treated with 2,4-D, ICA, IND and Trp as compared to the untreated 1021 cells. RT-PCR analysis was employed to analyze the differential expression patterns of nitrogen fixation genes under free-living and symbiotic conditions. Under symbiotic condition, the relative expression levels of nif and fix genes were significantly induced in Mt- RD64 plants and in Mt-1021 plants treated with IAA and 2,4-D whereas they were unchanged or repressed in Mt-1021 plants treated with the other selected compounds when compared to the untreated Mt-1021 plants. © 2010 Landes Bioscience

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging.

PubMed

Corydon, Thomas J; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-28

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24(th) DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31(st) parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.

Validation test of 125 Ah advanced design IPV nickel-hydrogen flight cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1993-01-01

An update of validation test results confirming the advanced design nickel-hydrogen cell is presented. An advanced 125 Ah individual pressure vessel Ni-H cell was designed. The primary function of the advanced cell is to store and deliver energy for long-term LEO spacecraft missions. The new features of this design are: (1) use of 26 percent rather than 31 percent KOH electrolyte; (2) use of a patented catalyzed wall wick; (3) use of serrated-edge separators to facilitate gaseous O and H flow within the cell, while maintaining physical contact with the wall wick for electrolyte management; and (4) use of a floating rather than a fixed stack to accommodate Ni electrode expansion due to charge/discharge cycling. The significant improvements resulting from these innovations are extended cycle life; enhanced thermal, electrolyte, and oxygen management; and accommodation of Ni electrode expansion. Six 125 Ah flight cells based on this design were fabricated; the catalyzed wall wick cells have been cycled for over 19,000 cycles with no cell failures in the continuing test. Two of the noncatalyzed wall wick cells failed (cycles 9588 and 13,900).

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging

PubMed Central

Corydon, Thomas J.; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-01

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization. PMID:26818711

Structure of the nucleoid in cells of Streptococcus faecalis.

PubMed Central

Daneo-Moore, L; Dicker, D; Higgins, M L

1980-01-01

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells. Images PMID:6767695

Results of the 2001 JPL Balloon Flight Solar Cell Calibration Program

NASA Technical Reports Server (NTRS)

Anspaugh, B. E.; Mueller, R. L.

2002-01-01

The 2001 solar cell calibration balloon flight campaign consisted of two flights, which occurred on June 26, 2001, and July 4, 2001. Fifty-nine modules were carried to an altitude of approximately 120,000 ft (36.6 km). Full I-V curves were measured on nineteen of these modules, and output at a fixed load was measured on thirty-two modules (forty-six cells), with some modules repeated on the second flight. Nine modules were flown for temperature measurement only. The data from the fixed load cells on the first flight was not usable. The temperature dependence of the first-flight data was erratic and we were unable to find a way to extract accurate calibration values. The I-V data from the first flight was good, however, and all data from the second flight was also good. The data was corrected to 28 C and to 1 AU (1.496 x 10(exp 8)km). The calibrated cells have been returned to their owners and can now be used as reference standards in simulator testing of cells and arrays.

Use of Different Furnaces to Study Repeatability and Reproducibility of Three Pd-C Cells

NASA Astrophysics Data System (ADS)

Battuello, M.; Florio, M.; Girard, F.

2010-09-01

Three different Pd-C eutectic fixed-point cells were prepared and investigated at INRIM. Several tens of phase transition runs were carried out and recorded with both a Si-based radiation thermometer at 950 nm and a precision InGaAs-based thermometer at 1.6 μm. Two of the cells were of the same design with an inner volume of 12 cm3. The third one was smaller with a useful inner volume of 3.6 cm3. The three cells were filled with palladium powder 4N5 or 4N8 pure and graphite powder 6N pure. The repeatability and stability of the inflection point were investigated over a period of 1 year. The noticeably different external dimensions of the two cells, namely, 110 mm and 40 mm in length, allowed the influence of the longitudinal temperature distribution to be investigated. For this purpose, two different furnaces, a single-zone with SiC heaters and a three-zone with MoSi2 heaters, were used. Different operative conditions, namely, temperature steps, melting rate, longitudinal temperature distributions, and position of cells within the furnace, were tested to investigate the reproducibility of the cells. Effects on the duration and shape of the plateaux were also studied. This article gives details of the measurement setup and analyses of the melting plateaux obtained with the different conditions.

Evaluation of a Treatment Approach Combining Nicotine Gum with Self-Guided Behavioral Treatments for Smoking Relapse Prevention.

ERIC Educational Resources Information Center

Killen, Joel D.; And Others

1990-01-01

Randomly assigned 1,218 smokers to cells in 4 (nicotine gum delivered ad lib, fixed regimen nicotine gum, placebo gum, no gum) x 3 (self-selected relapse prevention modules, randomly administered modules, no modules) design. Subjects receiving nicotine gum were more likely to be abstinent at 2- and 6-month followups. Fixed regimen accounted for…

ACVP-12: Quantitative Assessment of HIV/SIV Viral DNA in Laser Capture Microdissected (LCM) CD4+ T cell and/or Macrophage Populations from Formalin-Fixed Tissue Specimens | Frederick National Laboratory for Cancer Research

Cancer.gov

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. CD4 (DAB) and CD68/CD163 (FastRed) double immunohistochemistry will be performed, allowin

China Report, Science and Technology, White Paper, No. 1

DTIC Science & Technology

1987-04-02

traditional biotechnology to produce liquor, soy sauce, vinegar and other fermented food products. In the late fifties, China established an antibiotic...to transform the traditional fermentation industry, including the use of fixed fungi or fixed cells to make alcohol, beer, soy sauce, vinegar , and...use. We should also improve the techniques and equipment of fermentation , develop 35 the technologies of central heating and small-scale methane

Cortical microtubules in sweet clover columella cells developed in microgravity

NASA Technical Reports Server (NTRS)

Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1995-01-01

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.

Dynamic, mechanistic, molecular-level modelling of cyanobacteria: Anabaena and nitrogen interaction.

PubMed

Hellweger, Ferdi L; Fredrick, Neil D; McCarthy, Mark J; Gardner, Wayne S; Wilhelm, Steven W; Paerl, Hans W

2016-09-01

Phytoplankton (eutrophication, biogeochemical) models are important tools for ecosystem research and management, but they generally have not been updated to include modern biology. Here, we present a dynamic, mechanistic, molecular-level (i.e. gene, transcript, protein, metabolite) model of Anabaena - nitrogen interaction. The model was developed using the pattern-oriented approach to model definition and parameterization of complex agent-based models. It simulates individual filaments, each with individual cells, each with genes that are expressed to yield transcripts and proteins. Cells metabolize various forms of N, grow and divide, and differentiate heterocysts when fixed N is depleted. The model is informed by observations from 269 laboratory experiments from 55 papers published from 1942 to 2014. Within this database, we identified 331 emerging patterns, and, excluding inconsistencies in observations, the model reproduces 94% of them. To explore a practical application, we used the model to simulate nutrient reduction scenarios for a hypothetical lake. For a 50% N only loading reduction, the model predicts that N fixation increases, but this fixed N does not compensate for the loading reduction, and the chlorophyll a concentration decreases substantially (by 33%). When N is reduced along with P, the model predicts an additional 8% reduction (compared to P only). © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Slot angle detecting method for fiber fixed chip

NASA Astrophysics Data System (ADS)

Zhang, Jiaquan; Wang, Jiliang; Zhou, Chaochao

2018-04-01

The slot angle of fiber fixed chip has a significant impact on performance of photoelectric devices. In order to solve the actual engineering problem, this paper put forward a detecting method based on imaging processing. Because the images have very low contrast that is hardly segmented, so this paper proposes imaging segment methods based on edge character. Then get fixed chip edge line slope k2 and calculate the fiber fixed slot line slope k1, which can be used calculating the slot angle. Lastly, test the repeatability and accuracy of system, which show that this method has very fast operation speed and good robustness. Clearly, it is also satisfied to the actual demand of fiber fixed chip slot angle detection.

Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

PubMed Central

Limpens, Erik; Moling, Sjef; Hooiveld, Guido; Pereira, Patrícia A.; Bisseling, Ton; Becker, Jörg D.; Küster, Helge

2013-01-01

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies. PMID:23734198

New layer-based imaging and rapid prototyping techniques for computer-aided design and manufacture of custom dental restoration.

PubMed

Lee, M-Y; Chang, C-C; Ku, Y C

2008-01-01

Fixed dental restoration by conventional methods greatly relies on the skill and experience of the dental technician. The quality and accuracy of the final product depends mostly on the technician's subjective judgment. In addition, the traditional manual operation involves many complex procedures, and is a time-consuming and labour-intensive job. Most importantly, no quantitative design and manufacturing information is preserved for future retrieval. In this paper, a new device for scanning the dental profile and reconstructing 3D digital information of a dental model based on a layer-based imaging technique, called abrasive computer tomography (ACT) was designed in-house and proposed for the design of custom dental restoration. The fixed partial dental restoration was then produced by rapid prototyping (RP) and computer numerical control (CNC) machining methods based on the ACT scanned digital information. A force feedback sculptor (FreeForm system, Sensible Technologies, Inc., Cambridge MA, USA), which comprises 3D Touch technology, was applied to modify the morphology and design of the fixed dental restoration. In addition, a comparison of conventional manual operation and digital manufacture using both RP and CNC machining technologies for fixed dental restoration production is presented. Finally, a digital custom fixed restoration manufacturing protocol integrating proposed layer-based dental profile scanning, computer-aided design, 3D force feedback feature modification and advanced fixed restoration manufacturing techniques is illustrated. The proposed method provides solid evidence that computer-aided design and manufacturing technologies may become a new avenue for custom-made fixed restoration design, analysis, and production in the 21st century.

Donation return time at fixed and mobile donation sites

PubMed Central

Carey, Patricia M.; High, Patrick M.; Schlumpf, Karen S.; Johnson, Bryce R.; Mast, Alan E.; Rios, Jorge A.; Simon, Toby L.; Wilkinson, Susan L.

2013-01-01

BACKGROUND This study investigated the effect of blood donation environment, fixed or mobile with differing sponsor types, on donation return time. STUDY DESIGN AND METHODS Data from 2006 through 2009 at six US blood centers participating in the Retrovirus Epidemiology Donor Study-II (REDS-II) were used for analysis. Descriptive statistics stratified by whole blood (WB), plateletpheresis (PP), and double red blood cell (R2) donations were obtained for fixed and mobile locations, including median number of donations and median interdonation interval. A survival analysis estimated median return time at fixed and mobile sites, while controlling for censored return times, demographics, blood center, and mandatory recovery times. RESULTS Two-thirds (67.9%) of WB donations were made at mobile sites, 97.4% of PP donations were made at fixed sites, and R2 donations were equally distributed between fixed and mobile locations. For donations at fixed sites only or alternating between fixed and mobile sites, the highest median numbers of donations were nine and eight, respectively, and the shortest model-adjusted median return times (controlling for mandatory eligibility times of 56 and 112 days) were 36 and 30 days for WB and R2 donations, respectively. For PP donations, the shortest model-adjusted median return time was 23 days at a fixed location and the longest was 693 days at community locations. CONCLUSION WB, PP, and R2 donors with the shortest time between donations were associated with fixed locations and those alternating between fixed and mobile locations, even after controlling for differing mandatory recovery times for the different blood donation procedures. PMID:21745215

Pharmacokinetics, safety and efficacy of a recombinant factor IX product, trenonacog alfa in previously treated haemophilia B patients.

PubMed

Collins, P W; Quon, D V K; Makris, M; Chowdary, P; Kempton, C L; Apte, S J; Ramanan, M V; Hay, C R M; Drobic, B; Hua, Y; Babinchak, T J; Gomperts, E D

2018-01-01

Trenonacog alfa (IB1001) is a recombinant factor IX (rFIX) manufactured in Chinese hamster ovary (CHO) cells. IB1001 was evaluated in a multicentre clinical trial with haemophilia B patients. The aim was to establish IB1001 pharmacokinetic non-inferiority to comparator rFIX, safety and efficacy in previously treated patients (PTPs) with haemophilia B. Subjects were severe or moderately severe haemophilia B adult and adolescent PTPs with no history of FIX inhibitors. IB1001 PK non-inferiority to comparator rFIX was demonstrated through ratio of AUC 0-∞ in 32 subjects. IB1001 was well tolerated in all 76 treated subjects; the most common adverse drug reaction was headache (2.6% of subjects) and there were no reports of FIX inhibitors. Transient non-inhibitory binding FIX antibodies and anti-CHO cell protein antibodies developed in 21% and 29% of subjects respectively; no safety concerns were associated with development of these antibodies. Prophylaxis (mean duration ± SD: 17.9 ± 9.6 months, mean dose: 55.5 ± 12.9 IU/kg, median 1.0 infusion per week) was effective in preventing bleeds (median annual bleed rate: 1.52, interquartile range: 0.0-3.46). One or two IB1001 infusions resolved 84% of the bleeds, while for 84% of treatments haemostatic efficacy of IB1001 was rated excellent or good. IB1001 haemostatic efficacy for all 19 major surgeries was rated adequate or better than adequate. IB1001 is safe and efficacious for treatment of bleeds, routine prophylaxis and perioperative management in haemophilia B patients. © 2017 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

Effect of LEO cycling on 125 Ah advanced design IPV nickel-hydrogen battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

An advanced 125 Ah individual pressure vessel (IPV) nickel-hydrogen cell was designed. The primary function of the advanced cell, is to store and deliver energy for long term, low earth-orbit (LEO) spacecraft missions. The new features of this design are: (1) use of 26 percent rather than 31 percent potassium hydroxide (KOH) electrolyte, (2) use of a patented catalyzed wall wick, (3) use of serrated edge separators to facilitate gaseous oxygen and hydrogen flow within the cell, while still maintaining physical contact with the wall wick for electrolyte management, and (4) use of a floating rather than a fixed stack (state-of-the-art) to accommodate nickel electrode expansion. Six 125 Ah flight cells based on this design were fabricated by Eagle-Picher. Three of the cells contain all of the advanced features (test cells) and three are the same as the test cells except they don't have catalyst on the wall wick (control cells). All six cells are in the process of being evaluated in a LEO cycle life test. The cells have accumulated about 4700 LEO cycles (60 percent DOD 10 C). There have been no cell failures, the catalyzed wall wick cells however, are performing better.

Effect of LEO cycling on 125 Ah advanced design IPV nickel-hydrogen battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

An advanced 125 Ah individual pressure vessel (IPV) nickel-hydrogen cell was designed. The primary function of the advanced cell is to store and deliver energy for long-term, low earth-orbit (LEO) spacecraft missions. The new features of this design are: (1) use of 26 percent rather than 31 percent potassium hydroxide (KOH) electrolyte, (2) use of a patented catalyzed wall wick, (3) use of serrated-edge separators to facilitate gaseous oxygen and hydrogen flow within the cell, while still maintaining physical contact with the wall wick for electrolyte management, and (4) use of a floating rather than a fixed stack (state-of-the-art) to accommodate nickel electrode expansion. Six 125-Ah flight cells based on this design were fabricated by Eagle-Picher. Three of the cells contain all of the advanced features (test cells) and three are the same as the test cells except they don't have catalyst on the wall wick (control cells). All six cells are in the process of being evaluated in a LEO cycle life test. The cells have accumulated about 4700 LEO cycles (60 percent DOD 10 C). There have been no cell failures; the catalyzed wall wick cells, however, are performing better.

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

DOE PAGES

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry; ...

2016-08-31

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

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

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

PubMed Central

Veronese, S; Gambacorta, M; Falini, B

1989-01-01

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody. Images Fig 1 Fig 1 PMID:2475528

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

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.

PubMed

Wood, Henry M; Belvedere, Ornella; Conway, Caroline; Daly, Catherine; Chalkley, Rebecca; Bickerdike, Melissa; McKinley, Claire; Egan, Phil; Ross, Lisa; Hayward, Bruce; Morgan, Joanne; Davidson, Leslie; MacLennan, Ken; Ong, Thian K; Papagiannopoulos, Kostas; Cook, Ian; Adams, David J; Taylor, Graham R; Rabbitts, Pamela

2010-08-01

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

The Use of Variable Q1 Isolation Windows Improves Selectivity in LC-SWATH-MS Acquisition.

PubMed

Zhang, Ying; Bilbao, Aivett; Bruderer, Tobias; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard; Varesio, Emmanuel

2015-10-02

As tryptic peptides and metabolites are not equally distributed along the mass range, the probability of cross fragment ion interference is higher in certain windows when fixed Q1 SWATH windows are applied. We evaluated the benefits of utilizing variable Q1 SWATH windows with regards to selectivity improvement. Variable windows based on equalizing the distribution of either the precursor ion population (PIP) or the total ion current (TIC) within each window were generated by an in-house software, swathTUNER. These two variable Q1 SWATH window strategies outperformed, with respect to quantification and identification, the basic approach using a fixed window width (FIX) for proteomic profiling of human monocyte-derived dendritic cells (MDDCs). Thus, 13.8 and 8.4% additional peptide precursors, which resulted in 13.1 and 10.0% more proteins, were confidently identified by SWATH using the strategy PIP and TIC, respectively, in the MDDC proteomic sample. On the basis of the spectral library purity score, some improvement warranted by variable Q1 windows was also observed, albeit to a lesser extent, in the metabolomic profiling of human urine. We show that the novel concept of "scheduled SWATH" proposed here, which incorporates (i) variable isolation windows and (ii) precursor retention time segmentation further improves both peptide and metabolite identifications.

Zirconia based dental ceramics: structure, mechanical properties, biocompatibility and applications.

PubMed

Gautam, Chandkiram; Joyner, Jarin; Gautam, Amarendra; Rao, Jitendra; Vajtai, Robert

2016-12-06

Zirconia (ZrO 2 ) based dental ceramics have been considered to be advantageous materials with adequate mechanical properties for the manufacturing of medical devices. Due to its very high compression strength of 2000 MPa, ZrO 2 can resist differing mechanical environments. During the crack propagation on the application of stress on the surface of ZrO 2 , a crystalline modification diminishes the propagation of cracks. In addition, zirconia's biocompatibility has been studied in vivo, leading to the observation of no adverse response upon the insertion of ZrO 2 samples into the bone or muscle. In vitro experimentation has exhibited the absence of mutations and good viability of cells cultured on this material leading to the use of ZrO 2 in the manufacturing of hip head prostheses. The mechanical properties of zirconia fixed partial dentures (FPDs) have proven to be superior to other ceramic/composite restorations and hence leading to their significant applications in implant supported rehabilitations. Recent developments were focused on the synthesis of zirconia based dental materials. More recently, zirconia has been introduced in prosthetic dentistry for the fabrication of crowns and fixed partial dentures in combination with computer aided design/computer aided manufacturing (CAD/CAM) techniques. This systematic review covers the results of past as well as recent scientific studies on the properties of zirconia based ceramics such as their specific compositions, microstructures, mechanical strength, biocompatibility and other applications in dentistry.

COMBINING RATE-BASED AND CAP-AND-TRADE EMISSIONS POLICIES. (R828628)

EPA Science Inventory

Rate-based emissions policies (like tradable performance standards, TPS) fix average emissions intensity, while cap-and-trade (CAT) policies fix total emissions. This paper shows that unfettered trade between rate-based and cap-and-trade programs always raises combined emissio...

Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis

PubMed Central

Georgescu, Walter; Osseiran, Alma; Rojec, Maria; Liu, Yueyong; Bombrun, Maxime; Tang, Jonathan; Costes, Sylvain V.

2015-01-01

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation. PMID:26107175

Characterizing the DNA damage response by cell tracking algorithms and cell features classification using high-content time-lapse analysis

DOE PAGES

Georgescu, Walter; Osseiran, Alma; Rojec, Maria; ...

2015-06-24

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were ablemore » to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm 2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Furthermore, such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.« less

Improved Time-Lapsed Angular Scattering Microscopy of Single Cells

NASA Astrophysics Data System (ADS)

Cannaday, Ashley E.

By measuring angular scattering patterns from biological samples and fitting them with a Mie theory model, one can estimate the organelle size distribution within many cells. Quantitative organelle sizing of ensembles of cells using this method has been well established. Our goal is to develop the methodology to extend this approach to the single cell level, measuring the angular scattering at multiple time points and estimating the non-nuclear organelle size distribution parameters. The diameters of individual organelle-size beads were successfully extracted using scattering measurements with a minimum deflection angle of 20 degrees. However, the accuracy of size estimates can be limited by the angular range detected. In particular, simulations by our group suggest that, for cell organelle populations with a broader size distribution, the accuracy of size prediction improves substantially if the minimum angle of detection angle is 15 degrees or less. The system was therefore modified to collect scattering angles down to 10 degrees. To confirm experimentally that size predictions will become more stable when lower scattering angles are detected, initial validations were performed on individual polystyrene beads ranging in diameter from 1 to 5 microns. We found that the lower minimum angle enabled the width of this delta-function size distribution to be predicted more accurately. Scattering patterns were then acquired and analyzed from single mouse squamous cell carcinoma cells at multiple time points. The scattering patterns exhibit angular dependencies that look unlike those of any single sphere size, but are well-fit by a broad distribution of sizes, as expected. To determine the fluctuation level in the estimated size distribution due to measurement imperfections alone, formaldehyde-fixed cells were measured. Subsequent measurements on live (non-fixed) cells revealed an order of magnitude greater fluctuation in the estimated sizes compared to fixed cells. With our improved and better-understood approach to single cell angular scattering, we are now capable of reliably detecting changes in organelle size predictions due to biological causes above our measurement error of 20 nm, which enables us to apply our system to future studies of the investigation of various single cell biological processes.

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

PubMed Central

Cafferty, Patrick; Xie, Xiaojun; Browne, Kristen; Auld, Vanessa J.

2009-01-01

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals. PMID:19590493

The contribution of nitrogen fixation by cyanobacteria to particulate organic nitrogen in a constructed wetland

NASA Astrophysics Data System (ADS)

Zhang, X.; PAN, X.; MA, M.; Li, W.; Cui, L.

2016-12-01

N-fixing cyanobacteria can create extra nitrogen for aquatic ecosystems. Previous studies reported inconsistence patterns of the contribution of biological nitrogen fixation to the nitrogen pools in aquatic ecosystems. However, there were few studies concerning the effect of fixed nitrogen by cyanobacteria on the nitrogen removal efficiency in constructed wetlands. This study was performed at the Beijing Wildlife Rescue and Rehabilitation Centre, where a constructed lake for the habitation of waterfowls and a constructed wetland for purifying sewage from the lake are located. The composition of phytoplankton communities, the concentrations of particulate organic nitrogen (PON) and nitrogen fixation rates (Rn) in the constructed lake and the constructed wetland were compared throughout a growing season. We counted the densities of genus Anabaena and Microcystis cells, and explored their relationships with PON and Rn in water. The proportions of PON from various sources, including the ambient N2, waterfowl faeces, wetland sediments and the nitrates, were calculated by the natural abundance of 15N with the IsoSource software. The result revealed that the constructed lake was alternately dominated by Anabaena and Microcystis throughout the growing season, and the Rn was positively correlated with PON and the cell density of Anabaena (P < 0.05). This implied that the fixed nitrogen by N-fixing Anabaena might be utilized by non-N-fixing Microcystis, maintaining the fixed nitrogen with PON form. The ambient N2 composed 0.5 82% and 50.0 84.7% to the PON in the constructed lake and wetland respectively during the growing season. The proportions of PON from N2 increased to more than 80% when the Rn reached the highest in September. The result demonstrated that the nitrogen fixed by Anabaena might be utilized by non-N-fixing Microcystis which formed water blooms in summer. Therefore, the decline of the removal efficiency of PON in the constructed wetland in summer might indirectly result from the nitrogen fixation, since the proliferated algal were difficult to sediment in surface flow wetlands.

Infrared and Raman Microscopy in Cell Biology

PubMed Central

Matthäus, Christian; Bird, Benjamin; Miljković, Miloš; Chernenko, Tatyana; Romeo, Melissa; Diem, Max

2009-01-01

This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cell’s biochemical composition is collected at a spatial resolution of typically 25 mm. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes. PMID:19118679

IMMUNOREACTIONS INVOLVING PLATELETS

PubMed Central

Shulman, N. Raphael

1958-01-01

A steric and kinetic model for the sequence and mechanism of reactions leading to formation of a complex from an antibody, a haptene (quinidine), and a cell membrane (platelets), and to fixation of complement by the complex was deduced from the effects of varying the initial concentration of each component of the complex on the amount of complement fixed, from kinetic aspects of the sequential reactions, and from other chemical and physical properties of the various components involved. Theoretical results calculated using equations based on the model, which were derived by Dr. Terrell L. Hill, were similar in all respects to experimental results. Results of this study were consistent with the possibilities that the protein moiety of a haptenic antigen involved in development of an antibody which attaches to a cell is not necessarily a component of the cell, and that the cell reacts with the antibody by virtue of having a surface favorable for non-specific adsorption of certain haptene-antibody complexes. PMID:13525578

Extrapolation of radiation thermometry scales for determining the transition temperature of metal-carbon points. Experiments with the Co-C

NASA Astrophysics Data System (ADS)

Battuello, M.; Girard, F.; Florio, M.

2009-02-01

Four independent radiation temperature scales approximating the ITS-90 at 900 nm, 950 nm and 1.6 µm have been realized from the indium point (429.7485 K) to the copper point (1357.77 K) which were used to derive by extrapolation the transition temperature T90(Co-C) of the cobalt-carbon eutectic fixed point. An INRIM cell was investigated and an average value T90(Co-C) = 1597.20 K was found with the four values lying within 0.25 K. Alternatively, thermodynamic approximated scales were realized by assigning to the fixed points the best presently available thermodynamic values and deriving T(Co-C). An average value of 1597.27 K was found (four values lying within 0.25 K). The standard uncertainties associated with T90(Co-C) and T(Co-C) were 0.16 K and 0.17 K, respectively. INRIM determinations are compatible with recent thermodynamic determinations on three different cells (values lying between 1597.11 K and 1597.25 K) and with the result of a comparison on the same cell by an absolute radiation thermometer and an irradiance measurement with filter radiometers which give values of 1597.11 K and 1597.43 K, respectively (Anhalt et al 2006 Metrologia 43 S78-83). The INRIM approach allows the determination of both ITS-90 and thermodynamic temperature of a fixed point in a simple way and can provide valuable support to absolute radiometric methods in defining the transition temperature of new high-temperature fixed points.

Isolation of Mesophyll Cells and Bundle Sheath Cells from Digitaria sanguinalis (L.) Scop. Leaves and a Scanning Microscopy Study of the Internal Leaf Cell Morphology 1

PubMed Central

Edwards, Gerald E.; Black, Clanton C.

1971-01-01

A technique is described for the separation of mesophyll and bundle sheath cells from Digitaria sanguinalis leaves and evidence for separation is given with light and scanning electron micrographs. Gentle grinding of fully differentiated leaves in a mortar releases mesophyll cells which are isolated on nylon nets by filtration. More extensive grinding of the remaining tissue yields bundle sheath strands which are isolated by filtration with stainless steel sieves and nylon nets. Further grinding of bundle sheath strands in a tissue homogenizer releases bundle sheath cells which are collected on nylon nets. Percentage of purity derived from cell counts and yield data on a chlorophyll basis are given. The internal leaf cell morphology is presented in scanning electron micrographs and compared with light micrographs of fully-differentiated D. sanguinalis leaves. In leaves of plants which possess the C4-dicarboxylic acid cycle of photosynthesis, the relationship of leaf morphology to photosynthesis in mesophyll and bundle sheath cells is considered, and the hypothesis is presented that as atmospheric CO2 enters a leaf about 85% is fixed by the C4-dicarboxylic acid cycle in the mesophyll cells and 10 to 15% is fixed by the reductive pentose phosphate cycle in the bundle sheath cells. A technique also is given for the isolation of mesophyll cells from spinach leaves. Images PMID:16657571

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

Isolation of Mesophyll Cells and Bundle Sheath Cells from Digitaria sanguinalis (L.) Scop. Leaves and a Scanning Microscopy Study of the Internal Leaf Cell Morphology.

PubMed

Edwards, G E; Black, C C

1971-01-01

A technique is described for the separation of mesophyll and bundle sheath cells from Digitaria sanguinalis leaves and evidence for separation is given with light and scanning electron micrographs. Gentle grinding of fully differentiated leaves in a mortar releases mesophyll cells which are isolated on nylon nets by filtration. More extensive grinding of the remaining tissue yields bundle sheath strands which are isolated by filtration with stainless steel sieves and nylon nets. Further grinding of bundle sheath strands in a tissue homogenizer releases bundle sheath cells which are collected on nylon nets. Percentage of purity derived from cell counts and yield data on a chlorophyll basis are given.The internal leaf cell morphology is presented in scanning electron micrographs and compared with light micrographs of fully-differentiated D. sanguinalis leaves. In leaves of plants which possess the C(4)-dicarboxylic acid cycle of photosynthesis, the relationship of leaf morphology to photosynthesis in mesophyll and bundle sheath cells is considered, and the hypothesis is presented that as atmospheric CO(2) enters a leaf about 85% is fixed by the C(4)-dicarboxylic acid cycle in the mesophyll cells and 10 to 15% is fixed by the reductive pentose phosphate cycle in the bundle sheath cells.A technique also is given for the isolation of mesophyll cells from spinach leaves.

A simple reliable procedure for obtaining metaphases from human leukemic bone-marrow aspirates suitable for Giemsa banding.

PubMed

Srivastava, A K; Smith, R D

1980-02-01

Short incubation of heparinized human leukemic bone-marrow cells in phosphate buffered saline containing colcemid and overnight chilling of fixed cells yields metaphases with elongated and well-spread chromosomes. This technique enables us to do trypsin-Giemsa banding of chromosomes obtained from leukemic marrow cells otherwise difficult to band.

Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

USDA-ARS?s Scientific Manuscript database

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

PubMed Central

Thomas, W. Kelley; Vida, J. T.; Frisse, Linda M.; Mundo, Manuel; Baldwin, James G.

1997-01-01

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses. PMID:19274156

Expected Number of Fixed Points in Boolean Networks with Arbitrary Topology.

PubMed

Mori, Fumito; Mochizuki, Atsushi

2017-07-14

Boolean network models describe genetic, neural, and social dynamics in complex networks, where the dynamics depend generally on network topology. Fixed points in a genetic regulatory network are typically considered to correspond to cell types in an organism. We prove that the expected number of fixed points in a Boolean network, with Boolean functions drawn from probability distributions that are not required to be uniform or identical, is one, and is independent of network topology if only a feedback arc set satisfies a stochastic neutrality condition. We also demonstrate that the expected number is increased by the predominance of positive feedback in a cycle.

Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.

PubMed

Loudig, Olivier; Brandwein-Gensler, Margaret; Kim, Ryung S; Lin, Juan; Isayeva, Tatyana; Liu, Christina; Segall, Jeffrey E; Kenny, Paraic A; Prystowsky, Michael B

2011-12-01

High-throughput gene expression profiling from formalin-fixed, paraffin-embedded tissues has become a reality, and several methods are now commercially available. The Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay (Illumina, Inc) is a full-transcriptome version of the original 512-gene complementary DNA-mediated annealing, selection, extension and ligation assay, allowing high-throughput profiling of 24,526 annotated genes from degraded and formalin-fixed, paraffin-embedded RNA. This assay has the potential to allow identification of novel gene signatures associated with clinical outcome using banked archival pathology specimen resources. We tested the reproducibility of the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay and its sensitivity for detecting differentially expressed genes in RNA extracted from matched fresh and formalin-fixed, paraffin-embedded cells, after 1 and 13 months of storage, using the human breast cell lines MCF7 and MCF10A. Then, using tumor worst pattern of invasion as a classifier, 1 component of the "risk model," we selected 12 formalin-fixed, paraffin-embedded oral squamous cell carcinomas for whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay analysis. We profiled 5 tumors with nonaggressive, nondispersed pattern of invasion, and 7 tumors with aggressive dispersed pattern of invasion and satellites scattered at least 1 mm apart. To minimize variability, the formalin-fixed, paraffin-embedded specimens were prepared from snap-frozen tissues, and RNA was obtained within 24 hours of fixation. One hundred four down-regulated genes and 72 up-regulated genes in tumors with aggressive dispersed pattern of invasion were identified. We performed quantitative reverse transcriptase polymerase chain reaction validation of 4 genes using Taqman assays and in situ protein detection of 1 gene by immunohistochemistry. Functional cluster analysis of genes up-regulated in tumors with aggressive pattern of invasion suggests presence of genes involved in cellular cytoarchitecture, some of which already associated with tumor invasion. Identification of these genes provides biologic rationale for our histologic classification, with regard to tumor invasion, and demonstrates that the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay is a powerful assay for profiling degraded RNA from archived specimens when combined with quantitative reverse transcriptase polymerase chain reaction validation. Copyright © 2011 Elsevier Inc. All rights reserved.

47 CFR 24.5 - Terms and definitions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... in the National Geodetic Survey (NGS) data base. (Source: National Geodetic Survey, U.S. Department... antenna site. Base Station. A land station in the land mobile service. Broadband PCS. PCS services.... Fixed Station. A station in the fixed service. Land Mobile Service. A mobile service between base...

47 CFR 24.5 - Terms and definitions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... in the National Geodetic Survey (NGS) data base. (Source: National Geodetic Survey, U.S. Department... antenna site. Base Station. A land station in the land mobile service. Broadband PCS. PCS services.... Fixed Station. A station in the fixed service. Land Mobile Service. A mobile service between base...

47 CFR 24.5 - Terms and definitions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... in the National Geodetic Survey (NGS) data base. (Source: National Geodetic Survey, U.S. Department... antenna site. Base Station. A land station in the land mobile service. Broadband PCS. PCS services.... Fixed Station. A station in the fixed service. Land Mobile Service. A mobile service between base...

47 CFR 24.5 - Terms and definitions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... in the National Geodetic Survey (NGS) data base. (Source: National Geodetic Survey, U.S. Department... antenna site. Base Station. A land station in the land mobile service. Broadband PCS. PCS services.... Fixed Station. A station in the fixed service. Land Mobile Service. A mobile service between base...

Magnetoreception in birds: different physical processes for two types of directional responses

PubMed Central

Wiltschko, Roswitha; Stapput, Katrin; Ritz, Thorsten; Thalau, Peter; Wiltschko, Wolfgang

2007-01-01

Migratory orientation in birds involves an inclination compass based on radical-pair processes. Under certain light regimes, however, “fixed-direction” responses are observed that do not undergo the seasonal change between spring and autumn typical for migratory orientation. To identify the underlying transduction mechanisms, we analyzed a fixed-direction response under a combination of 502 nm turquoise and 590 nm yellow light, with migratory orientation under 565 nm green light serving as the control. High-frequency fields, diagnostic for a radical-pair mechanism, disrupted migratory orientation without affecting fixed-direction responses. Local anaesthesia of the upper beak where magnetite is found in birds, in contrast, disrupted the fixed-direction response without affecting migratory orientation. The two types of responses are thus based on different physical principles, with the compass response based on a radical pair mechanism and the fixed-direction responses probably originating in magnetite-based receptors in the upper beak. Directional input from these receptors seems to affect the behavior only when the regular inclination compass does not work properly. Evolutionary considerations suggest that magnetite-based receptors may represent an ancient mechanism that, in birds, has been replaced by the modern inclination compass based on radical-pair processes now used for directional orientation. PMID:19404459

Space-based pseudo-fixed latitude observation mode based on the characteristics of geosynchronous orbit belt

NASA Astrophysics Data System (ADS)

Hu, Yun-peng; Chen, Lei; Huang, Jian-yu

2017-08-01

The US Lincoln Laboratory proved that space-based visible (SBV) observation is efficient to observe space objects, especially Geosynchronous Orbit (GEO) objects. After that, SBV observation plays an important role in the space surveillance. In this paper, a novel space-based observation mode is designed to observe all the GEO objects in a relatively short time. A low earth orbit (LEO) satellite, especially a dawn-dusk sun-synchronous orbit satellite, is useful for space-based observation. Thus, the observation mode for GEO objects is based on a dawn-dusk sun-synchronous orbit satellite. It is found that the Pinch Point (PP) regions proposed by the US Lincoln Laboratory are spreading based on the analysis of the evolution principles of GEO objects. As the PP regions becoming more and more widely in the future, many strategies based on it may not be efficient any more. Hence, the key point of the space-based observation strategy design for GEO objects should be emphasized on the whole GEO belt as far as possible. The pseudo-fixed latitude observation mode is proposed in this paper based on the characteristics of GEO belt. Unlike classical space-based observation modes, pseudo-fixed latitude observation mode makes use of the one-dimensional attitude adjustment of the observation satellite. The pseudo-fixed latitude observation mode is more reliable and simple in engineering, compared with the gazing observation mode which needs to adjust the attitude from the two dimensions. It includes two types of attitude adjustment, i.e. daily and continuous attitude adjustment. Therefore, the pseudo-fixed latitude observation mode has two characteristics. In a day, the latitude of the observation region is fixed and the scanning region is about a rectangle, while the latitude of the observation region centre changes each day in a long term based on a daily strategy. The capabilities of a pseudo-fixed latitude observation instrument with a 98° dawn-dusk sun-synchronous orbit are discussed. It is found that most of GEO objects can be visited every day and almost all the GEO objects can be visited in two days in the whole year using a sensor with 20°×2° field of view (FOV). The seasonal drops, which are caused by the characteristics of GEO belt and the influence of earth shadow at the two equinoxes, have been overcome under the pseudo-fixed observation mode.

Organocatalytic removal of formaldehyde adducts from RNA and DNA bases.

PubMed

Karmakar, Saswata; Harcourt, Emily M; Hewings, David S; Scherer, Florian; Lovejoy, Alexander F; Kurtz, David M; Ehrenschwender, Thomas; Barandun, Luzi J; Roost, Caroline; Alizadeh, Ash A; Kool, Eric T

2015-09-01

Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases

PubMed Central

Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

2015-01-01

Formaldehyde is universally employed to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, and avoiding high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5–2.4 fold using a catalyst under optimized conditions, and by 7–25 fold compared to a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens. PMID:26291948

Non-scaling fixed field alternating gradient permanent magnet cancer therapy accelerator

DOEpatents

Trbojevic, Dejan

2017-05-23

A non-scaling fixed field alternating gradient accelerator includes a racetrack shape including a first straight section connected to a first arc section, the first arc section connected to a second straight section, the second straight section connected to a second arc section, and the second arc section connected to the first straight section; an matching cells configured to match particle orbits between the first straight section, the first arc section, the second straight section, and the second arc section. The accelerator includes the matching cells and an associated matching procedure enabling the particle orbits at varying energies between an arc section and a straight section in the racetrack shape.

Prosthetic Rehabilitation After Fibular Free Flap Surgery of Mandibular Defects in a Patient With Oral Squamous Cell Carcinoma.

PubMed

Yoon, Hyung-In

2016-10-01

This report is to present the treatment procedure and clinical considerations of prosthodontic management of a patient who had undergone a partial mandibulectomy and fibular free flap surgery. A 59-year-old man with a squamous cell carcinoma received a partial mandibular resection. Microsurgical reconstruction with a fibular free flap surgery and implant-supported zirconia-fixed prosthesis produced by computer-aided manufacturing led to successful results for the oral rehabilitation of mandibular defects. The implant-supported zirconia-fixed prosthesis can be recommended for use in patients with mandibulectomy and fibular free flaps. Close cooperation between the surgeon and the prosthodontist is mandatory for the satisfaction of the patient.

In-situ diagnostic tools for hydrogen transfer leak characterization in PEM fuel cell stacks part II: Operational applications

NASA Astrophysics Data System (ADS)

Niroumand, Amir M.; Homayouni, Hooman; DeVaal, Jake; Golnaraghi, Farid; Kjeang, Erik

2016-08-01

This paper describes a diagnostic tool for in-situ characterization of the rate and distribution of hydrogen transfer leaks in Polymer Electrolyte Membrane (PEM) fuel cell stacks. The method is based on reducing the air flow rate from a high to low value at a fixed current, while maintaining an anode overpressure. At high air flow rates, the reduction in air flow results in lower oxygen concentration in the cathode and therefore reduction in cell voltages. Once the air flow rate in each cell reaches a low value at which the cell oxygen-starves, the voltage of the corresponding cell drops to zero. However, oxygen starvation results from two processes: 1) the electrochemical oxygen reduction reaction which produces current; and 2) the chemical reaction between oxygen and the crossed over hydrogen. In this work, a diagnostic technique has been developed that accounts for the effect of the electrochemical reaction on cell voltage to identify the hydrogen leak rate and number of leaky cells in a fuel cell stack. This technique is suitable for leak characterization during fuel cell operation, as it only requires stack air flow and voltage measurements, which are readily available in an operational fuel cell system.

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

PubMed Central

Riddle, G D; Simonson, J G; Hales, B J; Braymer, H D

1982-01-01

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo. Images PMID:6956567

A cell-based study on pedestrian acceleration and overtaking in a transfer station corridor

NASA Astrophysics Data System (ADS)

Ji, Xiangfeng; Zhou, Xuemei; Ran, Bin

2013-04-01

Pedestrian speed in a transfer station corridor is faster than usual and sometimes running can be found among some of them. In this paper, pedestrians are divided into two categories. The first one is aggressive, and the other is conservative. Aggressive pedestrians weaving their way through crowd in the corridor are the study object of this paper. During recent decades, much attention has been paid to the pedestrians' behavior, such as overtaking (also deceleration) and collision avoidance, and that continues in this paper. After sufficiently analyzing the characteristics of pedestrian flow in transfer station corridor, a cell-based model is presented in this paper, including the acceleration (also deceleration) and overtaking analysis. Acceleration (also deceleration) in a corridor is fixed according to Newton's Law and then speed calculated with a kinematic formula is discretized into cells based on the fuzzy logic. After the speed is updated, overtaking is analyzed based on updated speed and force explicitly, compared to rule-based models, which herein we call implicit ones. During the analysis of overtaking, a threshold value to determine the overtaking direction is introduced. Actually, model in this paper is a two-step one. The first step is to update speed, which is the cells the pedestrian can move in one time interval and the other is to analyze the overtaking. Finally, a comparison between the rule-based cellular automata, the model in this paper and data in HCM 2000 is made to demonstrate our model can be used to achieve reasonable simulation of acceleration (also deceleration) and overtaking among pedestrians.

Development of a High-Throughput Ultrasound Technique for the Analysis of Tissue Engineering Constructs

PubMed Central

Stukel, Jessica; Goss, Monika; Zhou, Haoyan; Zhou, Wenda; Willits, Rebecca; Exner, Agata A.

2015-01-01

Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90± 2.58*10−5 a.u. compared to non-fiber forming intensity at 2.74± 0.36*10−5 a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5 ± 0.8*10−5 a.u. in 1 day live cells compared to 2.26 ± 0.39*10−5 a.u. in fixed cells at a concentration of 1*106 cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings. PMID:26577255

Development of a High-Throughput Ultrasound Technique for the Analysis of Tissue Engineering Constructs.

PubMed

Stukel, Jessica M; Goss, Monika; Zhou, Haoyan; Zhou, Wenda; Willits, Rebecca Kuntz; Exner, Agata A

2016-03-01

Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90 ± 2.58 × 10(-5) a.u. compared to non-fiber forming intensity at 2.74 ± 0.36 × 10(-5) a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5.00 ± 0.80 × 10(-5) a.u. in 1 day live cells compared to 2.26 ± 0.39 × 10(-5) a.u.in fixed cells at a concentration of 1 × 10(6) cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings.

First ultrastructural observations on gastritis caused by Physaloptera clausa (Spirurida: Physalopteridae) in hedgehogs (Erinaceus europeaus).

PubMed

Gorgani-Firouzjaee, T; Farshid, A A; Naem, S

2015-10-01

Ultrastructural changes of gastritis due to infection with Physaloptera clausa in 12 fresh carcasses of euthanized European hedgehogs (Erinaceus europaeus) collected from different part of Urmia, Iran, in which they were highly populated with this animal, six females and six males were subjected to detail necropsy with special reference to the stomach. Macroscopic changes of stomach were recorded and some of the worms collected. Based on number of parasites present in the stomach, they were divided into light infection, mild infection, and severe infection. Parasites were collected, and worms identification of the species was confirmed on the basis of light microscope examination with reference to keys. Tissues fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide and processed and plastic embedded; ultrathin sections of 60-70 nm were cut and stained with uranyl acetate and lead citrate; electron microscopic observations showed that, in light infection some changes were observed in gastric cells such as dilatation and vesiculation of the endoplasmic reticulum, large numbers of lipid granules, mitochondrial swelling, nuclear chromatin margination, and some nucleus showed washed out appearance. Other cells showed some alterations in mitochondria, dilatation of smooth endoplasmic reticulum, loss of both free and bound ribosomes, vesiculation in cytoplasm, and increase Golgi apparatus and secretory vesicles. The inflammatory cells including lymphocytes, macrophages, mast cells, and predominantly eosinophils were identified. In moderate infection, the cellular pattern of gastric mucosa replaced with inflammatory cells. The marked increase of macrophages and other inflammatory cell was observed. A particular finding in our study was the presence of globule leukocyte in the moderate stage. Moreover, scant formation and distribution of collagen fibers as well as fibroblasts were also noted. In severe infection, the most obvious observation was marked distribution of collagen fibers around the mucosal cells. The fibroblastic cells with elongated nucleus and extensive indentation were noticed. In conclusion, the result of our study revealed P. clausa could be a cause of gastritis and according to cellular pattern of inflammatory reaction, with the increase of worm burden and development of infection, chronic gastritis was stabilized. Present investigation documented the ultrastructural changes during verminous gastritis in hedgehogs.

Optical micro resonance based sensor schemes for detection and identification of nano particles and biological agents in situ

NASA Astrophysics Data System (ADS)

Saetchnikov, Vladimir A.; Tcherniavskaia, Elina A.; Schweiger, Gustav; Ostendorf, Andreas

2010-05-01

A novel emerging technique for the label-free analysis of nano particles including biomolecules using optical micro cavity resonance is being developed. Various schemes based on a mechanically fixed microspheres as well as microspheres melted by laser on the tip of a standard single mode fiber have been investigated to make further development for microbial application. Water solutions of ethanol, HCl, glucose, vitamin C and biotin have been used to test refractive index changes by monitoring the magnitude of the whispering gallery modes spectral shift. Particular efforts were made for effective fixing of the micro spheres in the water flow, an optimal geometry for micro resonance observation and material of microsphere the most appropriate for microbial application. Optical resonance in free micro spheres from PMMA fixed in micro channels produced by photolithography has been observed under the laser power of less then 1 microwatt. Resonance shifts of C reactive protein water solutions as well as albumin solutions in pure water and with HCl modelling blood have been investigated. Introducing controlled amount of glass gel nano particles into sensor microsphere surrounding were accompanied by both correlative resonance shift (400 nm in diameter) and total reconstruct of resonance spectra (57 nm in diameter). Developed schemes have been demonstrated to be a promising technology platform for sensitive, lab-on-chip type sensor of diagnostic tools for different biological molecules, e.g. proteins, oligonucleotides, oligosaccharides, lipids, small molecules, viral particles, cells as well as in different experimental contexts e.g. proteomics, genomics, drug discovery, and membrane studies.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

PubMed

Harris, Katherine E; Aldred, Shelley Force; Davison, Laura M; Ogana, Heather Anne N; Boudreau, Andrew; Brüggemann, Marianne; Osborn, Michael; Ma, Biao; Buelow, Benjamin; Clarke, Starlynn C; Dang, Kevin H; Iyer, Suhasini; Jorgensen, Brett; Pham, Duy T; Pratap, Payal P; Rangaswamy, Udaya S; Schellenberger, Ute; van Schooten, Wim C; Ugamraj, Harshad S; Vafa, Omid; Buelow, Roland; Trinklein, Nathan D

2018-01-01

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Phylogeny of Immune Recognition: Processing and Presentation of Structurally Defined Proteins in Channel Catfish Immune Responses

PubMed Central

Vallejo, Abbe N.; Miller, Norman W.

1991-01-01

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cell subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104. PMID:1668258

Dynamic imaging of protein-protein interactions by MP-FLIM

NASA Astrophysics Data System (ADS)

Ameer-Beg, Simon M.; Peter, Marion; Keppler, Melanie D.; Prag, Soren; Barber, Paul R.; Ng, Tony C.; Vojnovic, Borivoj

2005-03-01

The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.

N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species

USGS Publications Warehouse

Bazylinski, D.A.; Dean, A.J.; Schuler, D.; Phillips, E.J.P.; Lovley, D.R.

2000-01-01

Cells of Geobacter metallireducens, Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N2-dependent growth, the first anaerobically with Fe(lll) as the electron acceptor, and the latter three species micro-aerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N2 under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5 ?? 5.9nmol C2H4 produced min-1 mg-1 cell protein) while growing with Fe(lll) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum, indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum, fix atmospheric dinitrogen.

Electrokinetics of diffuse soft interfaces. 1. Limit of low Donnan potentials.

PubMed

Duval, Jérôme F L; van Leeuwen, Herman P

2004-11-09

The current theoretical approaches to electrokinetics of gels or polyelectrolyte layers are based on the assumption that the position of the very interface between the aqueous medium and the gel phase is well defined. Within this assumption, spatial profiles for the volume fraction of polymer segments (phi), the density of fixed charges in the porous layer (rho fix), and the coefficient modeling the friction to hydrodynamic flow (k) follow a step-function. In reality, the "fuzzy" nature of the charged soft layer is intrinsically incompatible with the concept of a sharp interface and therefore necessarily calls for more detailed spatial representations for phi, rho fix, and k. In this paper, the notion of diffuse interface is introduced. For the sake of illustration, linear spatial distributions for phi and rho fix are considered in the interfacial zone between the bulk of the porous charged layer and the bulk electrolyte solution. The corresponding distribution for k is inferred from the Brinkman equation, which for low phi reduces to Stokes' equation. Linear electrostatics, hydrodynamics, and electroosmosis issues are analytically solved within the context of streaming current and streaming potential of charged surface layers in a thin-layer cell. The hydrodynamic analysis clearly demonstrates the physical incorrectness of the concept of a discrete slip plane for diffuse interfaces. For moderate to low electrolyte concentrations and nanoscale spatial transition of phi from zero (bulk electrolyte) to phi o (bulk gel), the electrokinetic properties of the soft layer as predicted by the theory considerably deviate from those calculated on the basis of the discontinuous approximation by Ohshima.

The developmental basis for germline mosaicism in mouse and Drosophila melanogaster.

PubMed

Drost, J B; Lee, W R

1998-01-01

Data involving germline mosaics in Drosophila melanogaster and mouse are reconciled with developmental observations. Mutations that become fixed in the early embryo before separation of soma from the germline may, by the sampling process of development, continue as part of germline and/or differentiate into any somatic tissue. The cuticle of adult D. melanogaster, because of segmental development, can be used to estimate the proportion of mutant nuclei in the early embryo, but most somatic tissues and the germlines of both species continue from samples too small to be representative of the early embryo. Because of the small sample of cells/nuclei that remain in the germline after separation of soma in both species, mosaic germlines have percentages of mutant cells that vary widely, with a mean of 50% and an unusual platykurtic, flat-topped distribution. While the sampling process leads to similar statistical results for both species, their patterns of development are very different. In D. melanogaster the first differentiation is the separation of soma from germline with the germline continuing from a sample of only two to four nuclei, whereas the adult cuticle is a representative sample of cleavage nuclei. The presence of mosaicism in D. melanogaster germline is independent of mosaicism in the eye, head, and thorax. This independence was used to determine that mutations can occur at any of the early embryonic cell divisions and still average 50% mutant germ cells when the germline is mosaic; however, the later the mutation occurs, the higher the proportion of completely nonmutant germlines. In contrast to D. melanogaster, the first differentiation in the mouse does not separate soma from germline but produces the inner cell mass that is representative of the cleavage nuclei. Following formation of the primitive streak, the primordial germ cells develop at the base of the allantois and among a clonally related sample of cells, providing the same statistical distribution in the mouse germlines as in D. melanogaster. The proportion of mutations that are fixed during early embryonic development is greatly underestimated. For example, a DNA lesion in a postmeiotic gamete that becomes fixed as a dominant mutation during early embryonic development of the F1 may produce an individual completely mutant in the germ line and relevant somatic tissue or, alternatively, the F1 germline may be completely mutant but with no relevant somatic tissues for detecting the mutation until the F2. In both cases the mutation would be classified as complete in the F1 and F2, respectively, and not recognized as embryonic in origin. Because germ cells differentiate later in mammalian development, there are more opportunities for correlation between germline and soma in the mammal than Drosophila. However, because the germ cells and any somatic tissue, like blood, are derived from small samples, there may be many individuals that test negative in blood but have germlines that are either mosaic or entirely mutant.

Mathematical model to predict drivers' reaction speeds.

PubMed

Long, Benjamin L; Gillespie, A Isabella; Tanaka, Martin L

2012-02-01

Mental distractions and physical impairments can increase the risk of accidents by affecting a driver's ability to control the vehicle. In this article, we developed a linear mathematical model that can be used to quantitatively predict drivers' performance over a variety of possible driving conditions. Predictions were not limited only to conditions tested, but also included linear combinations of these tests conditions. Two groups of 12 participants were evaluated using a custom drivers' reaction speed testing device to evaluate the effect of cell phone talking, texting, and a fixed knee brace on the components of drivers' reaction speed. Cognitive reaction time was found to increase by 24% for cell phone talking and 74% for texting. The fixed knee brace increased musculoskeletal reaction time by 24%. These experimental data were used to develop a mathematical model to predict reaction speed for an untested condition, talking on a cell phone with a fixed knee brace. The model was verified by comparing the predicted reaction speed to measured experimental values from an independent test. The model predicted full braking time within 3% of the measured value. Although only a few influential conditions were evaluated, we present a general approach that can be expanded to include other types of distractions, impairments, and environmental conditions.

Light coupling into the Whispering Gallery Modes of a fiber array thin film solar cell for fixed partial Sun tracking

PubMed Central

Mariano, Marina; Rodríguez, Francisco J.; Romero-Gomez, Pablo; Kozyreff, Gregory; Martorell, Jordi

2014-01-01

We propose the use of whispering gallery mode coupling in a novel configuration based on implementing a thin film cell on the backside of an array of parallel fibers. We performed numerical calculations using the parameters of a thin film organic cell which demonstrate that light coupling becomes more effective as the angle for the incident light relative to the fiber array normal increases up to an optimal angle close to 55 deg. At this angle the power conversion efficiency of the fiber array solar cell we propose becomes 30% times larger than the one from an equivalent planar cell configuration. We demonstrate that the micro fiber array solar cell we propose may perform an effective partial tracking of the sun movement for over 100 degrees without any mechanical help. In addition, in the event that such fiber array cell would be installed with the adequate orientation on a vertical façade, an optimal photon-to-charge conversion would be reached for sunlight incident at 55 deg with respect to the horizon line, very close to the yearly average position for the sun at Latitude of 40 deg.

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.

PubMed

Hirose, H; Suhara, T; Kaji, N; Sakuma, N; Sekijima, M; Nojima, T; Miyakoshi, J

2008-01-01

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation. (c) 2007 Wiley-Liss, Inc.

49 CFR 611.301 - Small Starts eligibility.

Code of Federal Regulations, 2014 CFR

2014-10-01

... extension to a fixed guideway, or a corridor-based bus rapid transit system, a project must: (1) Be a Small... extension to a fixed guideway, or a corridor-based bus rapid system, a project must: (1) Be a Small Starts...

49 CFR 611.301 - Small Starts eligibility.

Code of Federal Regulations, 2013 CFR

2013-10-01

... extension to a fixed guideway, or a corridor-based bus rapid transit system, a project must: (1) Be a Small... extension to a fixed guideway, or a corridor-based bus rapid system, a project must: (1) Be a Small Starts...

A phase field approach for multicellular aggregate fusion in biofabrication.

PubMed

Yang, Xiaofeng; Sun, Yi; Wang, Qi

2013-07-01

We present a modeling and computational approach to study fusion of multicellular aggregates during tissue and organ fabrication, which forms the foundation for the scaffold-less biofabrication of tissues and organs known as bioprinting. It is known as the phase field method, where multicellular aggregates are modeled as mixtures of multiphase complex fluids whose phase mixing or separation is governed by interphase force interactions, mimicking the cell-cell interaction in the multicellular aggregates, and intermediate range interaction mediated by the surrounding hydrogel. The material transport in the mixture is dictated by hydrodynamics as well as forces due to the interphase interactions. In a multicellular aggregate system with fixed number of cells and fixed amount of the hydrogel medium, the effect of cell differentiation, proliferation, and death are neglected in the current model, which can be readily included in the model, and the interaction between different components is dictated by the interaction energy between cell and cell as well as between cell and medium particles, respectively. The modeling approach is applicable to transient simulations of fusion of cellular aggregate systems at the time and length scale appropriate to biofabrication. Numerical experiments are presented to demonstrate fusion and cell sorting during tissue and organ maturation processes in biofabrication.

Evidence-Based Design of Fixed-Dose Combinations: Principles and Application to Pediatric Anti-Tuberculosis Therapy.

PubMed

Svensson, Elin M; Yngman, Gunnar; Denti, Paolo; McIlleron, Helen; Kjellsson, Maria C; Karlsson, Mats O

2018-05-01

Fixed-dose combination formulations where several drugs are included in one tablet are important for the implementation of many long-term multidrug therapies. The selection of optimal dose ratios and tablet content of a fixed-dose combination and the design of individualized dosing regimens is a complex task, requiring multiple simultaneous considerations. In this work, a methodology for the rational design of a fixed-dose combination was developed and applied to the case of a three-drug pediatric anti-tuberculosis formulation individualized on body weight. The optimization methodology synthesizes information about the intended use population, the pharmacokinetic properties of the drugs, therapeutic targets, and practical constraints. A utility function is included to penalize deviations from the targets; a sequential estimation procedure was developed for stable estimation of break-points for individualized dosing. The suggested optimized pediatric anti-tuberculosis fixed-dose combination was compared with the recently launched World Health Organization-endorsed formulation. The optimized fixed-dose combination included 15, 36, and 16% higher amounts of rifampicin, isoniazid, and pyrazinamide, respectively. The optimized fixed-dose combination is expected to result in overall less deviation from the therapeutic targets based on adult exposure and substantially fewer children with underexposure (below half the target). The development of this design tool can aid the implementation of evidence-based formulations, integrating available knowledge and practical considerations, to optimize drug exposures and thereby treatment outcomes.

Nondestructive evaluation of epoxy-coated reinforcing bars in concrete using bi-electrode half-cell potential techniques.

DOT National Transportation Integrated Search

2006-01-01

This study evaluated two half-cell mapping methods for nondestructive evaluation of epoxy-coated rebar (ECR) in concrete: the semi-fixed bi-electrode and the moving bi-electrode methods. These methods were expected to provide early detection of corro...

Multi-color localization microscopy of fixed cells as a promising tool to study organization of bacterial cytoskeleton

NASA Astrophysics Data System (ADS)

Vedyaykin, A. D.; Gorbunov, V. V.; Sabantsev, A. V.; Polinovskaya, V. S.; Vishnyakov, I. E.; Melnikov, A. S.; Serdobintsev, P. Yu; Khodorkovskii, M. A.

2015-11-01

Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. Localization microscopy was used to study FtsZ organization in Escherichia coli previously in combination with fluorescent protein labeling, but the fact that fluorescent chimeric protein was unable to rescue temperature-sensitive ftsZ mutants suggests that obtained images may not represent native FtsZ structures faithfully. Indirect immunolabeling of FtsZ not only overcomes this problem, but also allows the use of the powerful visualization methods arsenal available for different structures in fixed cells. In this work we simultaneously obtained super-resolution images of FtsZ structures and diffraction-limited or super-resolution images of DNA and cell surface in E. coli, which allows for the study of the spatial arrangement of FtsZ structures with respect to the nucleoid positions and septum formation.

Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information.

PubMed

Koelsche, Christian; Hartmann, Wolfgang; Schrimpf, Daniel; Stichel, Damian; Jabar, Susanne; Ranft, Andreas; Reuss, David E; Sahm, Felix; Jones, David T W; Bewerunge-Hudler, Melanie; Trautmann, Marcel; Klingebiel, Thomas; Vokuhl, Christian; Gessler, Manfred; Wardelmann, Eva; Petersen, Iver; Baumhoer, Daniel; Flucke, Uta; Antonescu, Cristina; Esteller, Manel; Fröhling, Stefan; Kool, Marcel; Pfister, Stefan M; Mechtersheimer, Gunhild; Dirksen, Uta; von Deimling, Andreas

2018-03-23

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

PHYSIOLOGY OF NITROGEN FIXATION BY BACILLUS POLYMYXA

PubMed Central

Grau, F. H.; Wilson, P. W.

1962-01-01

Grau, F. H. (University of Wisconsin, Madison) and P. W. Wilson. Physiology of nitrogen fixation by Bacillus polymyxa. J. Bacteriol. 83:490–496. 1962.—Of 17 strains of Bacillus polymyxa tested for fixation of molecular nitrogen, 15 fixed considerable quantities (30 to 150 μg N/ml). Two strains of the closely related B. macerans did not use N2, but possibly other members of this species may do so. Confirmation of fixation was obtained by showing incorporation of N15 into cell material. Both iron and molybdenum are specifically required for fixation; without the addition of these metals to the nitrogen-free medium, the growth rate and the total nitrogen fixed were reduced about 30 to 50%. No requirement for added molybdenum could be shown when ammonia was the nitrogen source, and the absence of iron caused only a slight decrease in growth. Washed-cell suspensions of B. polymyxa containing an active hydrogenase readily incorporated N15 into cell materials when provided with mannitol, glucose, or pyruvate but not when formate was the substrate. Hydrogen is a specific inhibitor of fixation, reducing both the rate and final amount of nitrogen fixed; it did not reduce growth on ammonia. Fixation was strictly anaerobic, 1% oxygen in the gas phase being sufficient to stop fixation. Arsenate is a powerful inhibitor of fixation of N2 by washed-cell suspensions of B. polymyxa, indicating that high-energy phosphate may be significant for this process. PMID:13901244

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Sheep erythrocytes sensitized with amboceptor and persensitized thereafter with guinea pig complement are agglutinated by rabbit anti-guinea pig globulin and by immune sera obtained by injection of rabbits with fixed complement. In this agglutination neither C'1 nor C'2 takes part. Fixed C'4 acts as an agglutinogen. An additional agglutinogen, distinct from C'4, was found on persensitized cells. This additional agglutinogen appears to be distinct from hemolytically active C'3. PMID:14409703

Modular assembly of a photovoltaic solar energy receiver

DOEpatents

Graven, Robert M.; Gorski, Anthony J.; Schertz, William W.; Graae, Johan E. A.

1978-01-01

There is provided a modular assembly of a solar energy concentrator having a photovoltaic energy receiver with passive cooling. Solar cell means are fixedly coupled to a radiant energy concentrator. Tension means bias a large area heat sink against the cell thereby allowing the cell to expand or contract with respect to the heat sink due to differential heat expansion.

Results of continuous synchronous orbit testing of sealed nickel-cadmium cells

NASA Technical Reports Server (NTRS)

Harkness, J. D.

1981-01-01

Test results from continuous synchronous orbit testing of sealed nickel cadmium cells are presented. The synchronous orbit regime simulates a space satellite maintaining a position over a fixed point on earth as the earth rotates on its axis and revolves about the sun. Characteristics of each lot of cells, test conditions, and charge control methods are described.

Inverse size scaling of the nucleolus by a concentration-dependent phase transition.

PubMed

Weber, Stephanie C; Brangwynne, Clifford P

2015-03-02

Just as organ size typically increases with body size, the size of intracellular structures changes as cells grow and divide. Indeed, many organelles, such as the nucleus [1, 2], mitochondria [3], mitotic spindle [4, 5], and centrosome [6], exhibit size scaling, a phenomenon in which organelle size depends linearly on cell size. However, the mechanisms of organelle size scaling remain unclear. Here, we show that the size of the nucleolus, a membraneless organelle important for cell-size homeostasis [7], is coupled to cell size by an intracellular phase transition. We find that nucleolar size directly scales with cell size in early C. elegans embryos. Surprisingly, however, when embryo size is altered, we observe inverse scaling: nucleolar size increases in small cells and decreases in large cells. We demonstrate that this seemingly contradictory result arises from maternal loading of a fixed number rather than a fixed concentration of nucleolar components, which condense into nucleoli only above a threshold concentration. Our results suggest that the physics of phase transitions can dictate whether an organelle assembles, and, if so, its size, providing a mechanistic link between organelle assembly and cell size. Since the nucleolus is known to play a key role in cell growth, this biophysical readout of cell size could provide a novel feedback mechanism for growth control. Copyright © 2015 Elsevier Ltd. All rights reserved.

Three-dimensional cytomorphology in fine needle aspiration biopsy of medullary thyroid carcinoma.

PubMed

Chang, T C; Lai, S M; Wen, C Y; Hsiao, Y L; Huang, S H

2001-01-01

To elucidate three-dimensional (3-D) cytomorphology in fine needle aspiration biopsy (FNAB) of medullary thyroid carcinoma (MTC). ENAB was performed on tumors from five patients with MTC. The aspirate was stained and observed under a light microscope (LM). The aspirate was also fixed, dehydrated, critical point dried, spattered with gold ions and observed with a scanning electron microscope (SEM). For transmission electron microscopy (TEM), the specimen was fixed, dehydrated, embedded in an Epon mixture, cut with an ultramicrotome, mounted on copper grids, electron doubly stained with uranium acetate and lead citrate, and observed with TEM. Findings under SEM were correlated with those under LM and TEM. Under SEM, 3-D cytomorphology of MTC displayed a disorganized cellular arrangement with indistinct cell borders in three cases. The cell surface was uneven and had granular protrusions that corresponded to secretory granules observed under TEM. In one case with multiple endocrine neoplasia type IIB, there were abundant granules on the cell surface. In one case of sporadic MTC with multinucleated tumor giant cells and small cells, granular protrusions also were noted on the cell surface. Granular protrusion was a characteristic finding in FNAB of MTC tinder SEM and might be helpful in the differential diagnosis.

STS-26 MS Nelson on fixed based (FB) shuttle mission simulator (SMS) middeck

NASA Technical Reports Server (NTRS)

1988-01-01

STS-26 Discovery, Orbiter Vehicle (OV) 103, Mission Specialist (MS) George D. Nelson trains on the middeck of the fixed based (FB) shuttle mission simulator (SMS). Nelson, wearing communications assembly headset, adjusts camera mounting bracket.

47 CFR 90.745 - Phase I licensee service areas.

Code of Federal Regulations, 2010 CFR

2010-10-01

... be defined by the predicted 38 dBu service contour of its authorized base station or fixed station... its license to relocate its initially authorized base station. The Phase I licensee's predicted 38 dBu...'s base station or fixed station. Phase I licensees are permitted to add, remove, or modify...

Incident Involving 30-Ah Li-ion Cell at NASA Glenn Research Center

NASA Technical Reports Server (NTRS)

Bennett, William

2006-01-01

The key lesson learned from the February 17, 2006 cell explosion incident is that PC-based test-systems, even those having built-in watchdog monitors, can lose control and malfunction. In the case of lithiumion cell/battery testing, the stored energy can be released explosively causing considerable injury and damage to facilities. The investigation showed that although the Arbin system has a built-in watchdog monitor, the circumstances of the incident defeated the action of the watchdog and allowed the cycler to continue operation without control. An upgrade to the most recent version of Arbin software (version 4) was provided as a fix to the presumed control problem. This upgrade included newer EPROM s for the cycler microprocessor. Investigation revealed that similar incidents have occurred at other NASA centers with a variety of PC-based test instruments. JPL suffered an incident with Maccor testers and the GRC fuel cell group observed similar problems with LabView software. This is not exclusively an Arbin problem, but an issue with all PC-based systems. In this incident, it was fortunate that the event occurred after-hours with no-one in the room. The facility arrangement placed control consoles adjacent to the test chamber doors. Had someone been in the room during the event, they would have been exposed to hot debris and toxic combustion products. It was also fortunate that the exploded cell stayed inside the chamber after the door was forced open. If the cell had been ejected into the room it could have caused serious facility damage by impact and possibly caused a fire in the facility.

When Is Rapid On-Site Evaluation Cost-Effective for Fine-Needle Aspiration Biopsy?

PubMed Central

Schmidt, Robert L.; Walker, Brandon S.; Cohen, Michael B.

2015-01-01

Background Rapid on-site evaluation (ROSE) can improve adequacy rates of fine-needle aspiration biopsy (FNAB) but increases operational costs. The performance of ROSE relative to fixed sampling depends on many factors. It is not clear when ROSE is less costly than sampling with a fixed number of needle passes. The objective of this study was to determine the conditions under which ROSE is less costly than fixed sampling. Methods Cost comparison of sampling with and without ROSE using mathematical modeling. Models were based on a societal perspective and used a mechanistic, micro-costing approach. Sampling policies (ROSE, fixed) were compared using the difference in total expected costs per case. Scenarios were based on procedure complexity (palpation-guided or image-guided), adequacy rates (low, high) and sampling protocols (stopping criteria for ROSE and fixed sampling). One-way, probabilistic, and scenario-based sensitivity analysis was performed to determine which variables had the greatest influence on the cost difference. Results ROSE is favored relative to fixed sampling under the following conditions: (1) the cytologist is accurate, (2) the total variable cost ($/hr) is low, (3) fixed costs ($/procedure) are high, (4) the setup time is long, (5) the time between needle passes for ROSE is low, (6) when the per-pass adequacy rate is low, and (7) ROSE stops after observing one adequate sample. The model is most sensitive to variation in the fixed cost, the per-pass adequacy rate, and the time per needle pass with ROSE. Conclusions Mathematical modeling can be used to predict the difference in cost between sampling with and without ROSE. PMID:26317785

A threshold-based fixed predictor for JPEG-LS image compression

NASA Astrophysics Data System (ADS)

Deng, Lihua; Huang, Zhenghua; Yao, Shoukui

2018-03-01

In JPEG-LS, fixed predictor based on median edge detector (MED) only detect horizontal and vertical edges, and thus produces large prediction errors in the locality of diagonal edges. In this paper, we propose a threshold-based edge detection scheme for the fixed predictor. The proposed scheme can detect not only the horizontal and vertical edges, but also diagonal edges. For some certain thresholds, the proposed scheme can be simplified to other existing schemes. So, it can also be regarded as the integration of these existing schemes. For a suitable threshold, the accuracy of horizontal and vertical edges detection is higher than the existing median edge detection in JPEG-LS. Thus, the proposed fixed predictor outperforms the existing JPEG-LS predictors for all images tested, while the complexity of the overall algorithm is maintained at a similar level.

Fluctuations of cell population in a colonic crypt

NASA Astrophysics Data System (ADS)

Pei, Qi-ming; Zhan, Xuan; Yang, Li-jian; Bao, Chun; Cao, Wei; Li, An-bang; Rozi, Anvar; Jia, Ya

2014-03-01

The number of stem cells in a colonic crypt is often very small, which leads to large intrinsic fluctuations in the cell population. Based on the model of cell population dynamics with linear feedback in a colonic crypt, we present a stochastic dynamics of the cell population [including stem cells (SCs), transit amplifying cells (TACs), and fully differentiated cells (FDCs)]. The Fano factor, covariance, and susceptibility formulas of the cell population around the steady state are derived by using the Langevin theory. In the range of physiologically reasonable parameter values, it is found that the stationary populations of TACs and FDCs exhibit an approximately threshold behavior as a function of the net growth rate of TACs, and the reproductions of TACs and FDCs can be classified into three regimens: controlled, crossover, and uncontrolled. With the increasing of the net growth rate of TACs, there is a maximum of the relative intrinsic fluctuations (i.e., the Fano factors) of TACs and FDCs in the crossover region. For a fixed differentiation rate and the net growth rate of SCs, the covariance of fluctuations between SCs and TACs has a maximum in the crossover region. However, the susceptibilities of both TACs and FDCs to the net growth rate of TACs have a minimum in the crossover region.

IB-LBM simulation on blood cell sorting with a micro-fence structure.

PubMed

Wei, Qiang; Xu, Yuan-Qing; Tian, Fang-bao; Gao, Tian-xin; Tang, Xiao-ying; Zu, Wen-Hong

2014-01-01

A size-based blood cell sorting model with a micro-fence structure is proposed in the frame of immersed boundary and lattice Boltzmann method (IB-LBM). The fluid dynamics is obtained by solving the discrete lattice Boltzmann equation, and the cells motion and deformation are handled by the immersed boundary method. A micro-fence consists of two parallel slope post rows which are adopted to separate red blood cells (RBCs) from white blood cells (WBCs), in which the cells to be separated are transported one after another by the flow into the passageway between the two post rows. Effected by the cross flow, RBCs are schemed to get through the pores of the nether post row since they are smaller and more deformable compared with WBCs. WBCs are required to move along the nether post row till they get out the micro-fence. Simulation results indicate that for a fix width of pores, the slope angle of the post row plays an important role in cell sorting. The cells mixture can not be separated properly in a small slope angle, while obvious blockages by WBCs will take place to disturb the continuous cell sorting in a big slope angle. As an optimal result, an adaptive slope angle is found to sort RBCs form WBCs correctly and continuously.

Novel metabolic attributes of the genus cyanothece, comprising a group of unicellular nitrogen-fixing Cyanothece.

PubMed

Bandyopadhyay, Anindita; Elvitigala, Thanura; Welsh, Eric; Stöckel, Jana; Liberton, Michelle; Min, Hongtao; Sherman, Louis A; Pakrasi, Himadri B

2011-01-01

The genus Cyanothece comprises unicellular cyanobacteria that are morphologically diverse and ecologically versatile. Studies over the last decade have established members of this genus to be important components of the marine ecosystem, contributing significantly to the nitrogen and carbon cycle. System-level studies of Cyanothece sp. ATCC 51142, a prototypic member of this group, revealed many interesting metabolic attributes. To identify the metabolic traits that define this class of cyanobacteria, five additional Cyanothece strains were sequenced to completion. The presence of a large, contiguous nitrogenase gene cluster and the ability to carry out aerobic nitrogen fixation distinguish Cyanothece as a genus of unicellular, aerobic nitrogen-fixing cyanobacteria. Cyanothece cells can create an anoxic intracellular environment at night, allowing oxygen-sensitive processes to take place in these oxygenic organisms. Large carbohydrate reserves accumulate in the cells during the day, ensuring sufficient energy for the processes that require the anoxic phase of the cells. Our study indicates that this genus maintains a plastic genome, incorporating new metabolic capabilities while simultaneously retaining archaic metabolic traits, a unique combination which provides the flexibility to adapt to various ecological and environmental conditions. Rearrangement of the nitrogenase cluster in Cyanothece sp. strain 7425 and the concomitant loss of its aerobic nitrogen-fixing ability suggest that a similar mechanism might have been at play in cyanobacterial strains that eventually lost their nitrogen-fixing ability. The unicellular cyanobacterial genus Cyanothece has significant roles in the nitrogen cycle in aquatic and terrestrial environments. Cyanothece sp. ATCC 51142 was extensively studied over the last decade and has emerged as an important model photosynthetic microbe for bioenergy production. To expand our understanding of the distinctive metabolic capabilities of this cyanobacterial group, we analyzed the genome sequences of five additional Cyanothece strains from different geographical habitats, exhibiting diverse morphological and physiological attributes. These strains exhibit high rates of N(2) fixation and H(2) production under aerobic conditions. They can generate copious amounts of carbohydrates that are stored in large starch-like granules and facilitate energy-intensive processes during the dark, anoxic phase of the cells. The genomes of some Cyanothece strains are quite unique in that there are linear elements in addition to a large circular chromosome. Our study provides novel insights into the metabolism of this class of unicellular nitrogen-fixing cyanobacteria.

Influence of optic disc size on the diagnostic performance of macular ganglion cell complex and peripapillary retinal nerve fiber layer analyses in glaucoma.

PubMed

Cordeiro, Daniela Valença; Lima, Verônica Castro; Castro, Dinorah P; Castro, Leonardo C; Pacheco, Maria Angélica; Lee, Jae Min; Dimantas, Marcelo I; Prata, Tiago Santos

2011-01-01

To evaluate the influence of optic disc size on the diagnostic accuracy of macular ganglion cell complex (GCC) and conventional peripapillary retinal nerve fiber layer (pRNFL) analyses provided by spectral domain optical coherence tomography (SD-OCT) in glaucoma. Eighty-two glaucoma patients and 30 healthy subjects were included. All patients underwent GCC (7 × 7 mm macular grid, consisting of RNFL, ganglion cell and inner plexiform layers) and pRNFL thickness measurement (3.45 mm circular scan) by SD-OCT. One eye was randomly selected for analysis. Initially, receiver operating characteristic (ROC) curves were generated for different GCC and pRNFL parameters. The effect of disc area on the diagnostic accuracy of these parameters was evaluated using a logistic ROC regression model. Subsequently, 1.5, 2.0, and 2.5 mm(2) disc sizes were arbitrarily chosen (based on data distribution) and the predicted areas under the ROC curves (AUCs) and sensitivities were compared at fixed specificities for each. Average mean deviation index for glaucomatous eyes was -5.3 ± 5.2 dB. Similar AUCs were found for the best pRNFL (average thickness = 0.872) and GCC parameters (average thickness = 0.824; P = 0.19). The coefficient representing disc area in the ROC regression model was not statistically significant for average pRNFL thickness (-0.176) or average GCC thickness (0.088; P ≥ 0.56). AUCs for fixed disc areas (1.5, 2.0, and 2.5 mm(2)) were 0.904, 0.891, and 0.875 for average pRNFL thickness and 0.834, 0.842, and 0.851 for average GCC thickness, respectively. The highest sensitivities - at 80% specificity for average pRNFL (84.5%) and GCC thicknesses (74.5%) - were found with disc sizes fixed at 1.5 mm(2) and 2.5 mm(2). Diagnostic accuracy was similar between pRNFL and GCC thickness parameters. Although not statistically significant, there was a trend for a better diagnostic accuracy of pRNFL thickness measurement in cases of smaller discs. For GCC analysis, an inverse effect was observed.

A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation.

PubMed

Frithiof, Henrik; Aaltonen, Kristina; Rydén, Lisa

2016-01-01

Amplification of the HER-2/neu ( HER-2 ) proto-oncogene occurs in 10%-15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples. Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4',6-diamidino-2'-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs. A method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease. HER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments.

Isolation and Characterization of Prostate Cancer Stem Cells

DTIC Science & Technology

2013-10-01

et al. Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol 2011;8:97–106. 26] Guise T. Examining the...Nitrogen or fixed in formalin and paraffin-embedded to evaluate anatomy and glandular architecture. The remainder of the tissue was mechan- ically and

Light-Gated Memristor with Integrated Logic and Memory Functions.

PubMed

Tan, Hongwei; Liu, Gang; Yang, Huali; Yi, Xiaohui; Pan, Liang; Shang, Jie; Long, Shibing; Liu, Ming; Wu, Yihong; Li, Run-Wei

2017-11-28

Memristive devices are able to store and process information, which offers several key advantages over the transistor-based architectures. However, most of the two-terminal memristive devices have fixed functions once made and cannot be reconfigured for other situations. Here, we propose and demonstrate a memristive device "memlogic" (memory logic) as a nonvolatile switch of logic operations integrated with memory function in a single light-gated memristor. Based on nonvolatile light-modulated memristive switching behavior, a single memlogic cell is able to achieve optical and electrical mixed basic Boolean logic of reconfigurable "AND", "OR", and "NOT" operations. Furthermore, the single memlogic cell is also capable of functioning as an optical adder and digital-to-analog converter. All the memlogic outputs are memristive for in situ data storage due to the nonvolatile resistive switching and persistent photoconductivity effects. Thus, as a memdevice, the memlogic has potential for not only simplifying the programmable logic circuits but also building memristive multifunctional optoelectronics.

Morphodynamics of growing bacterial colony

NASA Astrophysics Data System (ADS)

Ghosh, Pushpita; Perlekar, Prasad; Rana, Navdeep

Self-organization into multicellular communities is a natural trend of most of the bacteria. Mutual interactions and competition among the bacterial cells in such multicellular organization play essential role in governing the spatiotemporal dynamics. We here present the spatiotemporal dynamics of growing bacterial colony using theory and a particle-based or individual-based simulation model of nonmotile cells growing utilizing a diffusing nutrient/food on a semi-solid surface by their growth and division forces and by pushing each-other through sliding motility. We show how the resource competition over a fixed amount of food, the diffusion coefficient of the nutrient and the random genetic noise govern the morphodynamics of a single species and a well-mixed two-species bacterial colonies. Our results show that for a very low initial food concentrations, colony develops fingering pattern at the front, while for intermediate values of initial food sources, the colony undergoes transitions to branched structures at the periphery and for very high values of food colony develops smoother fronts.

An approach for drag correction based on the local heterogeneity for gas-solid flows

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

Li, Tingwen; Wang, Limin; Rogers, William

2016-09-22

The drag models typically used for gas-solids interaction are mainly developed based on homogeneous systems of flow passing fixed particle assembly. It has been shown that the heterogeneous structures, i.e., clusters and bubbles in fluidized beds, need to be resolved to account for their effect in the numerical simulations. Since the heterogeneity is essentially captured through the local concentration gradient in the computational cells, this study proposes a simple approach to account for the non-uniformity of solids spatial distribution inside a computational cell and its effect on the interaction between gas and solid phases. Finally, to validate this approach, themore » predicted drag coefficient has been compared to the results from direct numerical simulations. In addition, the need to account for this type of heterogeneity is discussed for a periodic riser flow simulation with highly resolved numerical grids and the impact of the proposed correction for drag is demonstrated.« less

47 CFR 27.70 - Information exchange.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS... activated or an existing base or fixed station is modified: (1) Location; (2) Effective radiated power; (3... identify the source if interference is encountered when the base or fixed station is activated. [72 FR...

The Cellient System for Paraffin Histology Can Be Combined with HPV Testing and Morphotyping the Vaginal Microbiome Thanks to BoonFixing

PubMed Central

Boon, Mathilde E.

2013-01-01

The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides. PMID:23577033

The Cellient System for Paraffin Histology Can Be Combined with HPV Testing and Morphotyping the Vaginal Microbiome Thanks to BoonFixing.

PubMed

Boon, Mathilde E

2013-01-01

The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides.

Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia

NASA Astrophysics Data System (ADS)

Dekas, Anne E.; Poretsky, Rachel S.; Orphan, Victoria J.

2009-10-01

Nitrogen-fixing (diazotrophic) microorganisms regulate productivity in diverse ecosystems; however, the identities of diazotrophs are unknown in many oceanic environments. Using single-cell-resolution nanometer secondary ion mass spectrometry images of 15N incorporation, we showed that deep-sea anaerobic methane-oxidizing archaea fix N2, as well as structurally similar CN-, and share the products with sulfate-reducing bacterial symbionts. These archaeal/bacterial consortia are already recognized as the major sink of methane in benthic ecosystems, and we now identify them as a source of bioavailable nitrogen as well. The archaea maintain their methane oxidation rates while fixing N2 but reduce their growth, probably in compensation for the energetic burden of diazotrophy. This finding extends the demonstrated lower limits of respiratory energy capable of fueling N2 fixation and reveals a link between the global carbon, nitrogen, and sulfur cycles.

Genomic Instability and Breast Cancer

DTIC Science & Technology

2011-06-01

Survival Assay—Atotal of 1 103 cells were seeded onto a 60-mm dish in triplicate. Twenty-four hours after seeding, cells were irradiated by using a JL...ShepherdMark I-68A 137Cs- irradiator at indicated doses and incubated for 14 days. Result- ing colonies were fixed and stainedwithCoomassie Blue. Num...antibodies, cell culture, transfection and siRNAs, DNA substrates protein purification in insect cells, electrophoretic mobility shift assay and the ATPase

Experimental implementation of a biometric laser synaptic sensor.

PubMed

Pisarchik, Alexander N; Sevilla-Escoboza, Ricardo; Jaimes-Reátegui, Rider; Huerta-Cuellar, Guillermo; García-Lopez, J Hugo; Kazantsev, Victor B

2013-12-16

We fabricate a biometric laser fiber synaptic sensor to transmit information from one neuron cell to the other by an optical way. The optical synapse is constructed on the base of an erbium-doped fiber laser, whose pumped diode current is driven by a pre-synaptic FitzHugh-Nagumo electronic neuron, and the laser output controls a post-synaptic FitzHugh-Nagumo electronic neuron. The implemented laser synapse displays very rich dynamics, including fixed points, periodic orbits with different frequency-locking ratios and chaos. These regimes can be beneficial for efficient biorobotics, where behavioral flexibility subserved by synaptic connectivity is a challenge.

Speckle-field digital holographic microscopy.

PubMed

Park, YongKeun; Choi, Wonshik; Yaqoob, Zahid; Dasari, Ramachandra; Badizadegan, Kamran; Feld, Michael S

2009-07-20

The use of coherent light in conventional holographic phase microscopy (HPM) poses three major drawbacks: poor spatial resolution, weak depth sectioning, and fixed pattern noise due to unwanted diffraction. Here, we report a technique which can overcome these drawbacks, but maintains the advantage of phase microscopy - high contrast live cell imaging and 3D imaging. A speckle beam of a complex spatial pattern is used for illumination to reduce fixed pattern noise and to improve optical sectioning capability. By recording of the electric field of speckle, we demonstrate high contrast 3D live cell imaging without the need for axial scanning - neither objective lens nor sample stage. This technique has great potential in studying biological samples with improved sensitivity, resolution and optical sectioning capability.

Immunofluorescence detection of nitrogenase proteins in whole cells.

PubMed

Rennie, R J

1976-12-01

Fluorescent antibodies (FA) prepared against the Mo-Fe and Fe proteins of nitrogenase from Klebsiella pneumoniae M5aI were used to detect these protein components in toluene-treated whole cells that were actively reducing acetylene. The FA were highly specific, staining only nitrogenase component proteins originating from Klebsiella. Cross-reactions between the FA and purified nitrogenase proteins from other dinitrogen-fixing micro-organisms did not occur, except in the case of Bacillus polymyxa. The tests rapidly and accurately assayed the component proteins in Klebsiella mutants and derivatives to which Klebsiella nif genes had been transferred either by plasmid or by other means. Cross-reactions also indicated the degree of relatedness between nitrogenase proteins from dinitrogen-fixing micro-organisms of various origins.

Development of factors to convert frequency to rate for β-cell replication and apoptosis quantified by time-lapse video microscopy and immunohistochemistry

PubMed Central

Saisho, Yoshifumi; Manesso, Erica; Gurlo, Tatyana; Huang, Chang-jiang; Toffolo, Gianna M.; Cobelli, Claudio; Butler, Peter C.

2009-01-01

An obstacle to development of methods to quantify β-cell turnover from pancreas tissue is the lack of conversion factors for the frequency of β-cell replication or apoptosis detected by immunohistochemistry to rates of replication or apoptosis. We addressed this obstacle in islets from 1-mo-old rats by quantifying the relationship between the rate of β-cell replication observed directly by time-lapse video microscopy (TLVM) and the frequency of β-cell replication in the same islets detected by immunohistochemistry using antibodies against Ki67 and insulin in the same islets fixed immediately after TLVM. Similarly, we quantified the rate of β-cell apoptosis by TLVM and then the frequency of apoptosis in the same islets using TdT-mediated dUTP nick-end labeling and insulin. Conversion factors were developed by regression analysis. The conversion factor from Ki67 labeling frequency (%) to actual replication rate (%events/h) is 0.025 ± 0.003 h−1. The conversion factor from TdT-mediated dUTP nick-end labeling frequency (%) to actual apoptosis rate (%events/h) is 0.41 ± 0.05 h−1. These conversion factors will permit development of models to evaluate β-cell turnover in fixed pancreas tissue. PMID:18940937

"NEW MEMBRANE" FORMATION IN AMOEBA PROTEUS UPON INJURY OF INDIVIDUAL CELLS

PubMed Central

Szubinska, Barbara

1971-01-01

Changes in the plasma membrane complex following the injury of single cells of Amoeba proteus were examined with the electron microscope. Two types of injury were employed in this study; cells were either pinched ("cut") in half or speared with a glass microneedle, and quickly fixed. Speared cells, when fixed in the presence of the ruthenium violet (a derivative of ruthenium red), revealed the presence of an extra trilaminar structure outside of each cell. This structure, called the "new membrane," was separated from the plasma membrane complex by a distance of less than a micron. The trilaminar structure of the new membrane strikingly resembled the image of the plasma membrane in all cells examined, except for its increased width (30%). This new membrane appeared nearly to surround the injured amebae. Attempts were made to demonstrate the possible origin of the new membrane, its reality, and its sensitivity to calcium. Also, some evidence is shown concerning the role of the small dense droplets (100–1200 A in diameter) normally present in the cytoplasm of amebae. Their frequent contact with the plasma membrane of the cell as the result of injury is interpreted as indicating their involvement in the formation and expansion of the plasma membrane. PMID:4103955

RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.

PubMed

Gloghini, Annunziata; Canal, Barbara; Klein, Ulf; Dal Maso, Luigino; Perin, Tiziana; Dalla-Favera, Riccardo; Carbone, Antonino

2004-11-01

In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.

Information transmission on hybrid networks

NASA Astrophysics Data System (ADS)

Chen, Rongbin; Cui, Wei; Pu, Cunlai; Li, Jie; Ji, Bo; Gakis, Konstantinos; Pardalos, Panos M.

2018-01-01

Many real-world communication networks often have hybrid nature with both fixed nodes and moving modes, such as the mobile phone networks mainly composed of fixed base stations and mobile phones. In this paper, we discuss the information transmission process on the hybrid networks with both fixed and mobile nodes. The fixed nodes (base stations) are connected as a spatial lattice on the plane forming the information-carrying backbone, while the mobile nodes (users), which are the sources and destinations of information packets, connect to their current nearest fixed nodes respectively to deliver and receive information packets. We observe the phase transition of traffic load in the hybrid network when the packet generation rate goes from below and then above a critical value, which measures the network capacity of packets delivery. We obtain the optimal speed of moving nodes leading to the maximum network capacity. We further improve the network capacity by rewiring the fixed nodes and by considering the current load of fixed nodes during packets transmission. Our purpose is to optimize the network capacity of hybrid networks from the perspective of network science, and provide some insights for the construction of future communication infrastructures.

Mitochondrial imaging in live or fixed tissues using a luminescent iridium complex.

PubMed

Sorvina, Alexandra; Bader, Christie A; Darby, Jack R T; Lock, Mitchell C; Soo, Jia Yin; Johnson, Ian R D; Caporale, Chiara; Voelcker, Nicolas H; Stagni, Stefano; Massi, Massimiliano; Morrison, Janna L; Plush, Sally E; Brooks, Douglas A

2018-05-29

Mitochondrial morphology is important for the function of this critical organelle and, accordingly, altered mitochondrial structure is exhibited in many pathologies. Imaging of mitochondria can therefore provide important information about disease presence and progression. However, mitochondrial imaging is currently limited by the availability of agents that have the capacity to image mitochondrial morphology in both live and fixed samples. This can be particularly problematic in clinical studies or large, multi-centre cohort studies, where tissue archiving by fixation is often more practical. We previously reported the synthesis of an iridium coordination complex [Ir(ppy) 2 (MeTzPyPhCN)] + ; where ppy is a cyclometalated 2-phenylpyridine and TzPyPhCN is the 5-(5-(4-cyanophen-1-yl)pyrid-2-yl)tetrazolate ligand; and showed that this complex (herein referred to as IraZolve-Mito) has a high specificity for mitochondria in live cells. Here we demonstrate that IraZolve-Mito can also effectively stain mitochondria in both live and fixed tissue samples. The staining protocol proposed is versatile, providing a universal procedure for cell biologists and pathologists to visualise mitochondria.

Optofluidic solar concentrators using electrowetting tracking: Concept, design, and characterization

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

Cheng, JT; Park, S; Chen, CL

2013-03-01

We introduce a novel optofluidic solar concentration system based on electrowetting tracking. With two immiscible fluids in a transparent cell, we can actively control the orientation of fluid fluid interface via electrowetting. The naturally-formed meniscus between the two liquids can function as a dynamic optical prism for solar tracking and sunlight steering. An integrated optofluidic solar concentrator can be constructed from the liquid prism tracker in combination with a fixed and static optical condenser (Fresnel lens). Therefore, the liquid prisms can adaptively focus sunlight on a concentrating photovoltaic (CPV) cell sitting on the focus of the Fresnel lens as themore » sun moves. Because of the unique design, electrowetting tracking allows the concentrator to adaptively track both the daily and seasonal changes of the sun's orbit (dual-axis tracking) without bulky, expensive and inefficient mechanical moving parts. This approach can potentially reduce capital costs for CPV and increases operational efficiency by eliminating the power consumption of mechanical tracking. Importantly, the elimination of bulky tracking hardware and quiet operation will allow extensive residential deployment of concentrated solar power. In comparison with traditional silicon-based photovoltaic (PV) solar cells, the electrowetting-based self-tracking technology will generate,similar to 70% more green energy with a 50% cost reduction. (C) 2013 Elsevier Ltd. All rights reserved.« less

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a

PubMed Central

Jeyapalan, Jessie C.; Sedivy, John M.

2013-01-01

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5×107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems. PMID:23454889

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a.

PubMed

Jeyapalan, Jessie C; Sedivy, John M

2013-02-01

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5x107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.

Weather Impact on Airport Arrival Meter Fix Throughput

NASA Technical Reports Server (NTRS)

Wang, Yao

2017-01-01

Time-based flow management provides arrival aircraft schedules based on arrival airport conditions, airport capacity, required spacing, and weather conditions. In order to meet a scheduled time at which arrival aircraft can cross an airport arrival meter fix prior to entering the airport terminal airspace, air traffic controllers make regulations on air traffic. Severe weather may create an airport arrival bottleneck if one or more of airport arrival meter fixes are partially or completely blocked by the weather and the arrival demand has not been reduced accordingly. Under these conditions, aircraft are frequently being put in holding patterns until they can be rerouted. A model that predicts the weather impacted meter fix throughput may help air traffic controllers direct arrival flows into the airport more efficiently, minimizing arrival meter fix congestion. This paper presents an analysis of air traffic flows across arrival meter fixes at the Newark Liberty International Airport (EWR). Several scenarios of weather impacted EWR arrival fix flows are described. Furthermore, multiple linear regression and regression tree ensemble learning approaches for translating multiple sector Weather Impacted Traffic Indexes (WITI) to EWR arrival meter fix throughputs are examined. These weather translation models are developed and validated using the EWR arrival flight and weather data for the period of April-September in 2014. This study also compares the performance of the regression tree ensemble with traditional multiple linear regression models for estimating the weather impacted throughputs at each of the EWR arrival meter fixes. For all meter fixes investigated, the results from the regression tree ensemble weather translation models show a stronger correlation between model outputs and observed meter fix throughputs than that produced from multiple linear regression method.

The effect of fixed oil and water extracts of Nigella sativa on sickle cells: an in vitro study.

PubMed

Ibraheem, N K; Ahmed, J H; Hassan, M K

2010-03-01

Various drugs have been investigated in the treatment of sickle cell disease (SCD), such as hydroxyurea, piracetam and calcium antagonists. Most of these drugs are potentially toxic and are not suitable for long-term therapy. Recently, Nigella sativa (NS) has been reported to have calcium antagonist and antioxidant activities, both of which play a role in the management of the disease. This study aimed to investigate the in vitro antisickling effect of extracts from NS. Thirty-two patients with SCD, aged 7-47 years old, were recruited for the study. A total of 3 ml of venous blood was collected from each patient and divided into six tubes with heparin. The blood was mixed with 0.5 ml of either 0.1 percent, 0.05 percent or 0.01 percent v/v of the oil extract of NS. A slide was prepared by spreading a drop of treated blood, covered with a cover slide to ensure the complete deoxygenation condition. The separation of irreversibly sickled cells (ISCs) was performed on eight patients by a density gradient (Percoll-Renografin) centrifugation method. The 0.1 percent v/v concentration of the oil extract of NS resulted in an approximately 80 percent reduction in the formation of sickle cells. The 0.05 percent v/v concentration of NS produced an intermediate effect, while the 0.01 percent v/v concentration had no effect on the formation of sickle cells. The 0.1 percent v/v concentration of the fixed oil of NS led to a considerable reduction in the formation of ISCs. The fixed oil extracted from NS seeds has an in vitro antisickling activity.

Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

NASA Technical Reports Server (NTRS)

Chakrabarti, Amitabha; Stoecker, Andrew; Schatten, Heide

1995-01-01

To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods.

Biochemical component identification by plasmonic improved whispering gallery mode optical resonance based sensor

NASA Astrophysics Data System (ADS)

Saetchnikov, Vladimir A.; Tcherniavskaia, Elina A.; Saetchnikov, Anton V.; Schweiger, Gustav; Ostendorf, Andreas

2014-05-01

Experimental data on detection and identification of variety of biochemical agents, such as proteins, microelements, antibiotic of different generation etc. in both single and multi component solutions under varied in wide range concentration analyzed on the light scattering parameters of whispering gallery mode optical resonance based sensor are represented. Multiplexing on parameters and components has been realized using developed fluidic sensor cell with fixed in adhesive layer dielectric microspheres and data processing. Biochemical component identification has been performed by developed network analysis techniques. Developed approach is demonstrated to be applicable both for single agent and for multi component biochemical analysis. Novel technique based on optical resonance on microring structures, plasmon resonance and identification tools has been developed. To improve a sensitivity of microring structures microspheres fixed by adhesive had been treated previously by gold nanoparticle solution. Another technique used thin film gold layers deposited on the substrate below adhesive. Both biomolecule and nanoparticle injections caused considerable changes of optical resonance spectra. Plasmonic gold layers under optimized thickness also improve parameters of optical resonance spectra. Biochemical component identification has been also performed by developed network analysis techniques both for single and for multi component solution. So advantages of plasmon enhancing optical microcavity resonance with multiparameter identification tools is used for development of a new platform for ultra sensitive label-free biomedical sensor.

Lagrangian Modeling of Evaporating Sprays at Diesel Engine Conditions: Effects of Multi-Hole Injector Nozzles With JP-8 Surrogates

DTIC Science & Technology

2014-05-01

solver to treat the spray process. An Adaptive Mesh Refinement (AMR) and fixed embedding technique is employed to capture the gas - liquid interface with...Adaptive Mesh Refinement (AMR) and fixed embedding technique is employed to capture the gas - liquid interface with high fidelity while keeping the cell...in single and multi-hole nozzle configurations. The models were added to the present CONVERGE liquid fuel database and validated extensively

Novel Spectro-Temporal Codes and Computations for Auditory Signal Representation and Separation

DTIC Science & Technology

2013-02-01

responses are shown). Bottom right panel (c) shows the Frequency responses of the tunable bandpass filter ( BPF ) triplets that adapt to the incoming...signal. One BPF triplet is associated with each fixed filter, such that coarse filtering of the fixed gammatone filters is followed by additional, finer...is achieved using a second layer of narrower bandpass filters ( BPFs , Q=8) that emulate the filtering functions of outer hair cells (OHCs). In the

Three-Dimensional Viscous Flow Analysis for Moving Bodies Past Fixed Structures

DTIC Science & Technology

1988-05-13

BELLEVUE, WA 98n)05 Research Triangle Park, UC 27709-2211 6Sý. NAME Of FUNDING I PONSORING O Ib. C’FFICE SYMBOL 9 . PROCUREMENT INSTRUMENT IPENTIFICATION...34 otheor sditico Grs IMa ý; pl S- Three- Dimvensio:.iýal Viscrous Flow Analysis for Moving Bodies Past Fixed Structures Fina.11Report, Kelton M. Peery and...Recommendations 40 List of Figures 1 Finite-Volume Mesh ......... ......................... 8 2 Finite-Volume Cell ....... ............................ 9 3

Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

PubMed

Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás

2015-01-01

To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

PubMed Central

Chu, Kung-Hui; Alvarez-Cohen, Lisa

1998-01-01

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments. PMID:9726896

47 CFR 90.475 - Operation of internal transmitter control systems in specially equipped systems.

Code of Federal Regulations, 2010 CFR

2010-10-01

... systems involving fixed systems whose base stations are controlled by such systems may automatically access these base stations through the microwave or operational fixed systems from positions in the PSTN, so long as the base stations and mobile units meet the requirements of § 90.483 and if a separate...

Manipulation of cells' position across a microfluidic channel using a series of continuously varying herringbone structures

NASA Astrophysics Data System (ADS)

Jung, Yugyung; Hyun, Ji-chul; Choi, Jongchan; Atajanov, Arslan; Yang, Sung

2017-12-01

Controlling cells' movement is an important technique in biological analysis that is performed within a microfluidic system. Many external forces are utilized for manipulation of cells, including their position in the channel. These forces can effectively control cells in a desired manner. Most of techniques used to manipulate cells require sophisticated set-ups and equipment to generate desired effect. The exception to this is the use of hydrodynamic force. In this study, a series of continuously varying herringbone structures is proposed for positioning cells in a microfluidic channel using hydrodynamic force. This structure was experimentally developed by changing parameters, such as the length of the herringbone's apex, the length of the herringbone's base and the ratio of the height of the flat channel to the height of the herringbone structure. Results of this study, have demonstrated that the length of the herringbone's apex and the ratio of the heights of the flat channel and the herringbone structure were crucial parameters influencing positioning of cells at 100 μl/h flow rate. The final design was fixed at 170 and 80 μm for the length of herringbone's apex and the length of herringbone's base, respectively. The average position of cells in this device was 34 μm away from the side wall in a 200 μm wide channel. Finally, to substantiate a practical application of the herringbone structure for positioning, cells were randomly introduced into a microfluidic device, containing an array of trapping structures together with a series of herringbone structures along the channel. The cells were moved toward the trapping structure by the herringbone structure and the trapping efficiency was increased. Therefore, it is anticipated that this device will be utilized to continuously control cells' position without application of external forces.

Simulation of light-induced degradation of μc-Si in a-Si/μc-Si tandem solar cells by the diode equivalent circuit

NASA Astrophysics Data System (ADS)

Weicht, J. A.; Hamelmann, F. U.; Behrens, G.

2016-02-01

Silicon-based thin film tandem solar cells consist of one amorphous (a-Si) and one microcrystalline (μc-Si) silicon solar cell. The Staebler - Wronski effect describes the light- induced degradation and temperature-dependent healing of defects of silicon-based solar thin film cells. The solar cell degradation depends strongly on operation temperature. Until now, only the light-induced degradation (LID) of the amorphous layer was examined in a-Si/μc-Si solar cells. The LID is also observed in pc-Si single function solar cells. In our work we show the influence of the light-induced degradation of the μc-Si layer on the diode equivalent circuit. The current-voltage-curves (I-V-curves) for the initial state of a-Si/pc-Si modules are measured. Afterwards the cells are degraded under controlled conditions at constant temperature and constant irradiation. At fixed times the modules are measured at standard test conditions (STC) (AM1.5, 25°C cell temperature, 1000 W/m2) for controlling the status of LID. After the degradation the modules are annealed at dark conditions for several hours at 120°C. After the annealing the dangling bonds in the amorphous layer are healed, while the degradation of the pc-Si is still present, because the healing of defects in pc-Si solar cells needs longer time or higher temperatures. The solar cells are measured again at STC. With this laboratory measured I-V-curves we are able to separate the values of the diode model: series Rs and parallel resistance Rp, saturation current Is and diode factor n.

Biofunctionalized aligned microgels provide 3D cell guidance to mimic complex tissue matrices.

PubMed

Rose, Jonas C; Gehlen, David B; Haraszti, Tamás; Köhler, Jens; Licht, Christopher J; De Laporte, Laura

2018-05-01

Natural healing is based on highly orchestrated processes, in which the extracellular matrix plays a key role. To resemble the native cell environment, we introduce an artificial extracellular matrix (aECM) with the capability to template hierarchical and anisotropic structures in situ, allowing a minimally-invasive application via injection. Synthetic, magnetically responsive, rod-shaped microgels are locally aligned and fixed by a biocompatible surrounding hydrogel, creating a hybrid anisotropic hydrogel (Anisogel), of which the physical, mechanical, and chemical properties can be tailored. The microgels are rendered cell-adhesive with GRGDS and incorporated either inside a cell-adhesive fibrin or bioinert poly(ethylene glycol) hydrogel to strongly interact with fibroblasts. GRGDS-modified microgels inside a fibrin-based Anisogel enhance fibroblast alignment and lead to a reduction in fibronectin production, indicating successful replacement of structural proteins. In addition, YAP-translocation to the nucleus increases with the concentration of microgels, indicating cellular sensing of the overall anisotropic mechanical properties of the Anisogel. For bioinert surrounding PEG hydrogels, GRGDS-microgels are required to support cell proliferation and fibronectin production. In contrast to fibroblasts, primary nerve growth is not significantly affected by the biomodification of the microgels. In conclusion, this approach opens new opportunities towards advanced and complex aECMs for tissue regeneration. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

APMP Scale Comparison with Three Radiation Thermometers and Six Fixed-Point Blackbodies

NASA Astrophysics Data System (ADS)

Yamada, Y.; Shimizu, Y.; Ishii, J.

2015-08-01

New Asia Pacific Metrology Programme (APMP) comparisons of radiation thermometry standards, APMP TS-11, and -12, have recently been initiated. These new APMP comparisons cover the temperature range from to . Three radiation thermometers with central wavelengths of 1.6 , 0.9 , and 0.65 are the transfer devices for the radiation thermometer scale comparison conducted in the so-called star configuration. In parallel, a compact fixed-point blackbody furnace that houses six types of fixed-point cells of In, Sn, Zn, Al, Ag, and Cu is circulated, again in a star-type comparison, to substantiate fixed-point calibration capabilities. Twelve APMP national metrology institutes are taking part in this endeavor, in which the National Metrology Institute of Japan acts as the pilot. In this article, the comparison scheme is described with emphasis on the features of the transfer devices, i.e., the radiation thermometers and the fixed-point blackbodies. Results of preliminary evaluations of the performance and characteristic of these instruments as well as the evaluation method of the comparison results are presented.

Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells.

PubMed

Jessel, Robert; Haertel, Steffen; Socaciu, Carmen; Tykhonova, Svetlana; Diehl, Horst A

2002-01-01

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.

Fixed Point Learning Based Intelligent Traffic Control System

NASA Astrophysics Data System (ADS)

Zongyao, Wang; Cong, Sui; Cheng, Shao

2017-10-01

Fixed point learning has become an important tool to analyse large scale distributed system such as urban traffic network. This paper presents a fixed point learning based intelligence traffic network control system. The system applies convergence property of fixed point theorem to optimize the traffic flow density. The intelligence traffic control system achieves maximum road resources usage by averaging traffic flow density among the traffic network. The intelligence traffic network control system is built based on decentralized structure and intelligence cooperation. No central control is needed to manage the system. The proposed system is simple, effective and feasible for practical use. The performance of the system is tested via theoretical proof and simulations. The results demonstrate that the system can effectively solve the traffic congestion problem and increase the vehicles average speed. It also proves that the system is flexible, reliable and feasible for practical use.

An efficient algorithm for computing fixed length attractors based on bounded model checking in synchronous Boolean networks with biochemical applications.

PubMed

Li, X Y; Yang, G W; Zheng, D S; Guo, W S; Hung, W N N

2015-04-28

Genetic regulatory networks are the key to understanding biochemical systems. One condition of the genetic regulatory network under different living environments can be modeled as a synchronous Boolean network. The attractors of these Boolean networks will help biologists to identify determinant and stable factors. Existing methods identify attractors based on a random initial state or the entire state simultaneously. They cannot identify the fixed length attractors directly. The complexity of including time increases exponentially with respect to the attractor number and length of attractors. This study used the bounded model checking to quickly locate fixed length attractors. Based on the SAT solver, we propose a new algorithm for efficiently computing the fixed length attractors, which is more suitable for large Boolean networks and numerous attractors' networks. After comparison using the tool BooleNet, empirical experiments involving biochemical systems demonstrated the feasibility and efficiency of our approach.

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.

PubMed

Manno, Catherine S; Pierce, Glenn F; Arruda, Valder R; Glader, Bertil; Ragni, Margaret; Rasko, John J; Rasko, John; Ozelo, Margareth C; Hoots, Keith; Blatt, Philip; Konkle, Barbara; Dake, Michael; Kaye, Robin; Razavi, Mahmood; Zajko, Albert; Zehnder, James; Rustagi, Pradip K; Nakai, Hiroyuki; Chew, Amy; Leonard, Debra; Wright, J Fraser; Lessard, Ruth R; Sommer, Jürg M; Tigges, Michael; Sabatino, Denise; Luk, Alvin; Jiang, Haiyan; Mingozzi, Federico; Couto, Linda; Ertl, Hildegund C; High, Katherine A; Kay, Mark A

2006-03-01

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.

Passive participation of fixed platelets in aggregation facilitated by covalently bound fibrinogen.

PubMed

Agam, G; Livne, A

1983-01-01

The role of fibrinogen in interplatelet recognition during aggregation was examined by combining two cell types: fresh platelets (in limiting density) activated by thrombin or A23187, and formaldehyde-fixed platelets, bearing cross-linked fibrinogen. The fixed platelets did not aggregate by themselves, nor with resting platelets, but were capable of interacting with activated platelets and of participating passively in aggregation. The participation, expressed by enhanced aggregation, was assayed by the conventional turbidometric traces and by cosedimentation of fixed 3H-platelets with aggregates of fresh platelets. Platelet suspensions, prepared without special means to avert spontaneous activation, retained plasma fibrinogen to the extent of 50 micrograms/ml of a suspension containing 10(8) platelets, and the derived fixed platelets participated in aggregation, independently of added fibrinogen. The capability of such fixed platelets to participate in aggregation was sensitive to proteolytic digestion and to massive acetylation. When platelet separation was aided by apyrase or aspirin, PGE1 and gel filtration, the residual plasma fibrinogen was limited to 0.4 micrograms/ml of 10(8) platelet suspension. The derived fixed platelets were incapable of participating in aggregation unless fibrinogen was added prior to fixation. The affixed fibrinogen could not be replaced by soluble fibrinogen or affixed albumin. It is concluded that fibrinogen, which binds to platelets upon activation or is linked to them covalently, is a recognition site for platelet-platelet interaction during aggregation.

32 CFR 37.315 - What are the advantages of using a fixed-support TIA?

Code of Federal Regulations, 2010 CFR

2010-07-01

... DoD GRANT AND AGREEMENT REGULATIONS TECHNOLOGY INVESTMENT AGREEMENTS Expenditure-Based and Fixed-Support Technology Investment Agreements § 37.315 What are the advantages of using a fixed-support TIA? In... public accountants of the recipient's books and records. (d) Set minimum standards for the recipient's...

Entropy based quantification of Ki-67 positive cell images and its evaluation by a reader study

NASA Astrophysics Data System (ADS)

Niazi, M. Khalid Khan; Pennell, Michael; Elkins, Camille; Hemminger, Jessica; Jin, Ming; Kirby, Sean; Kurt, Habibe; Miller, Barrie; Plocharczyk, Elizabeth; Roth, Rachel; Ziegler, Rebecca; Shana'ah, Arwa; Racke, Fred; Lozanski, Gerard; Gurcan, Metin N.

2013-03-01

Presence of Ki-67, a nuclear protein, is typically used to measure cell proliferation. The quantification of the Ki-67 proliferation index is performed visually by the pathologist; however, this is subject to inter- and intra-reader variability. Automated techniques utilizing digital image analysis by computers have emerged. The large variations in specimen preparation, staining, and imaging as well as true biological heterogeneity of tumor tissue often results in variable intensities in Ki-67 stained images. These variations affect the performance of currently developed methods. To optimize the segmentation of Ki-67 stained cells, one should define a data dependent transformation that will account for these color variations instead of defining a fixed linear transformation to separate different hues. To address these issues in images of tissue stained with Ki-67, we propose a methodology that exploits the intrinsic properties of CIE L∗a∗b∗ color space to translate this complex problem into an automatic entropy based thresholding problem. The developed method was evaluated through two reader studies with pathology residents and expert hematopathologists. Agreement between the proposed method and the expert pathologists was good (CCC = 0.80).

An application of a Hill-based response surface model for a drug combination experiment on lung cancer.

PubMed

Ning, Shaoyang; Xu, Hongquan; Al-Shyoukh, Ibrahim; Feng, Jiaying; Sun, Ren

2014-10-30

Combination chemotherapy with multiple drugs has been widely applied to cancer treatment owing to enhanced efficacy and reduced drug resistance. For drug combination experiment analysis, response surface modeling has been commonly adopted. In this paper, we introduce a Hill-based global response surface model and provide an application of the model to a 512-run drug combination experiment with three chemicals, namely AG490, U0126, and indirubin-3  ' -monoxime (I-3-M), on lung cancer cells. The results demonstrate generally improved goodness of fit of our model from the traditional polynomial model, as well as the original Hill model on the basis of fixed-ratio drug combinations. We identify different dose-effect patterns between normal and cancer cells on the basis of our model, which indicates the potential effectiveness of the drug combination in cancer treatment. Meanwhile, drug interactions are analyzed both qualitatively and quantitatively. The distinct interaction patterns between U0126 and I-3-M on two types of cells uncovered by the model could be a further indicator of the efficacy of the drug combination. Copyright © 2014 John Wiley & Sons, Ltd.

Vertical ascending electrophoresis of cells with a minimal stabilizing medium

NASA Technical Reports Server (NTRS)

Omenyi, S. N.; Snyder, R. S.

1983-01-01

Vertical fractionation of a mixture of fixed horse and human red blood cells layered over a stabilizing support medium was done to give a valid comparison with proposed space experiments. In particular, the effects of sample thickness and concentration on zone migration rate were investigated. Electrophoretic mobilities of horse and human cells calculated from zone migration rates were compatible with those obtained by microelectrophoresis. Complete cell separation was observed when low power and effective cooling were employed.

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

PubMed Central

Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

2013-01-01

DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAF V600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used. PMID:23691506

Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

2016-04-01

The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

Noise power spectrum of the fixed pattern noise in digital radiography detectors

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

Kim, Dong Sik, E-mail: dskim@hufs.ac.kr; Kim, Eun

Purpose: The fixed pattern noise in radiography image detectors is caused by various sources. Multiple readout circuits with gate drivers and charge amplifiers are used to efficiently acquire the pixel voltage signals. However, the multiple circuits are not identical and thus yield nonuniform system gains. Nonuniform sensitivities are also produced from local variations in the charge collection elements. Furthermore, in phosphor-based detectors, the optical scattering at the top surface of the columnar CsI growth, the grain boundaries, and the disorder structure causes spatial sensitivity variations. These nonuniform gains or sensitivities cause fixed pattern noise and degrade the detector performance, evenmore » though the noise problem can be partially alleviated by using gain correction techniques. Hence, in order to develop good detectors, comparative analysis of the energy spectrum of the fixed pattern noise is important. Methods: In order to observe the energy spectrum of the fixed pattern noise, a normalized noise power spectrum (NNPS) of the fixed pattern noise is considered in this paper. Since the fixed pattern noise is mainly caused by the nonuniform gains, we call the spectrum the gain NNPS. We first asymptotically observe the gain NNPS and then formulate two relationships to calculate the gain NNPS based on a nonuniform-gain model. Since the gain NNPS values are quite low compared to the usual NNPS, measuring such a low NNPS value is difficult. By using the average of the uniform exposure images, a robust measuring method for the gain NNPS is proposed in this paper. Results: By using the proposed measuring method, the gain NNPS curves of several prototypes of general radiography and mammography detectors were measured to analyze their fixed pattern noise properties. We notice that a direct detector, which is based on the a-Se photoconductor, showed lower gain NNPS than the indirect-detector case, which is based on the CsI scintillator. By comparing the gain NNPS curves of the indirect detectors, we could analyze the scintillator properties depending on the techniques for the scintillator surface processing. Conclusions: A robust measuring method for the NNPS of the fixed pattern noise of a radiography detector is proposed in this paper. The method can measure a stable gain NNPS curve, even though the fixed pattern noise level is quite low. From the measured gain NNPS curves, we can compare and analyze the detector properties in terms of producing the fixed pattern noise.« less

Antibody-supervised deep learning for quantification of tumor-infiltrating immune cells in hematoxylin and eosin stained breast cancer samples.

PubMed

Turkki, Riku; Linder, Nina; Kovanen, Panu E; Pellinen, Teijo; Lundin, Johan

2016-01-01

Immune cell infiltration in tumor is an emerging prognostic biomarker in breast cancer. The gold standard for quantification of immune cells in tissue sections is visual assessment through a microscope, which is subjective and semi-quantitative. In this study, we propose and evaluate an approach based on antibody-guided annotation and deep learning to quantify immune cell-rich areas in hematoxylin and eosin (H&E) stained samples. Consecutive sections of formalin-fixed parafin-embedded samples obtained from the primary tumor of twenty breast cancer patients were cut and stained with H&E and the pan-leukocyte CD45 antibody. The stained slides were digitally scanned, and a training set of immune cell-rich and cell-poor tissue regions was annotated in H&E whole-slide images using the CD45-expression as a guide. In analysis, the images were divided into small homogenous regions, superpixels, from which features were extracted using a pretrained convolutional neural network (CNN) and classified with a support of vector machine. The CNN approach was compared to texture-based classification and to visual assessments performed by two pathologists. In a set of 123,442 labeled superpixels, the CNN approach achieved an F-score of 0.94 (range: 0.92-0.94) in discrimination of immune cell-rich and cell-poor regions, as compared to an F-score of 0.88 (range: 0.87-0.89) obtained with the texture-based classification. When compared to visual assessment of 200 images, an agreement of 90% (κ = 0.79) to quantify immune infiltration with the CNN approach was achieved while the inter-observer agreement between pathologists was 90% (κ = 0.78). Our findings indicate that deep learning can be applied to quantify immune cell infiltration in breast cancer samples using a basic morphology staining only. A good discrimination of immune cell-rich areas was achieved, well in concordance with both leukocyte antigen expression and pathologists' visual assessment.

Isolation of guard cells from fresh epidermis using a piezo-power micro-dissection system with vibration-attenuated needles.

PubMed

Terpitz, Ulrich; Zimmermann, Dirk

2010-01-01

The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

Optimization of self-aligned double patterning (SADP)-compliant layout designs using pattern matching for sub-20nm metal routing

NASA Astrophysics Data System (ADS)

Wang, Lynn T.-N.; Schroeder, Uwe Paul; Madhavan, Sriram

2017-03-01

A pattern-based methodology for optimizing SADP-compliant layout designs is developed based on identifying cut mask patterns and replacing them with pre-characterized fixing solutions. A pattern-based library of difficult-tomanufacture cut patterns with pre-characterized fixing solutions is built. A pattern-based engine searches for matching patterns in the decomposed layouts. When a match is found, the engine opportunistically replaces the detected pattern with a pre-characterized fixing solution. The methodology was demonstrated on a 7nm routed metal2 block. A small library of 30 cut patterns increased the number of more manufacturable cuts by 38% and metal-via enclosure by 13% with a small parasitic capacitance impact of 0.3%.

Empirical comparison of a fixed-base and a moving-base simulation of a helicopter engaged in visually conducted slalom runs

NASA Technical Reports Server (NTRS)

Parrish, R. V.; Houck, J. A.; Martin, D. J., Jr.

1977-01-01

Combined visual, motion, and aural cues for a helicopter engaged in visually conducted slalom runs at low altitude were studied. The evaluation of the visual and aural cues was subjective, whereas the motion cues were evaluated both subjectively and objectively. Subjective and objective results coincided in the area of control activity. Generally, less control activity is present under motion conditions than under fixed-base conditions, a fact attributed subjectively to the feeling of realistic limitations of a machine (helicopter) given by the addition of motion cues. The objective data also revealed that the slalom runs were conducted at significantly higher altitudes under motion conditions than under fixed-base conditions.

Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots

PubMed Central

Robledo, M.; Jiménez-Zurdo, J. I.; Velázquez, E.; Trujillo, M. E.; Zurdo-Piñeiro, J. L.; Ramírez-Bahena, M. H.; Ramos, B.; Díaz-Mínguez, J. M.; Dazzo, F.; Martínez-Molina, E.; Mateos, P. F.

2008-01-01

The rhizobia–legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis. PMID:18458328

Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B.

PubMed

Cao, Ou; Hoffman, Brad E; Moghimi, Babak; Nayak, Sushrusha; Cooper, Mario; Zhou, Shangzhen; Ertl, Hildegund C J; High, Katherine A; Herzog, Roland W

2009-10-01

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

The use of solar energy - photovoltaic - in hydrogen production and arid zones like Saudi Arabia

NASA Astrophysics Data System (ADS)

Sayigh, A. A. M.

This paper deals with the use of photovoltaic technology for the production of hydrogen from water by electrolysis. First of all the amount of electricity needed for this process was assessed, then various types of solar cell systems to generate the electricity needed were discussed and the best system was established. Some of the investigations involved testing of solar cells with concentrators and with fixed tilt or tracking devices. Several small panels of solar cells were used in testing the effect of local dust and sand as well as the fixed tilt in the area of Riyadh. The cost of producing hydrogen by electrolysis using electricity from a conventional grid was calculated. This cost was compared with the cost of production of hydrogen if a solar cell array was used. The paper outlines the continuous price increase of oil to produce electricity and the rapid decrease in price of solar cells. Both these advances will lead to a cheaper way of producing hydrogen by solar energy. In addition it is shown that technology is almost trouble free and requires very little know-how as far as operation is concerned.

Controlling Energy Radiations of Electromagnetic Waves via Frequency Coding Metamaterials.

PubMed

Wu, Haotian; Liu, Shuo; Wan, Xiang; Zhang, Lei; Wang, Dan; Li, Lianlin; Cui, Tie Jun

2017-09-01

Metamaterials are artificial structures composed of subwavelength unit cells to control electromagnetic (EM) waves. The spatial coding representation of metamaterial has the ability to describe the material in a digital way. The spatial coding metamaterials are typically constructed by unit cells that have similar shapes with fixed functionality. Here, the concept of frequency coding metamaterial is proposed, which achieves different controls of EM energy radiations with a fixed spatial coding pattern when the frequency changes. In this case, not only different phase responses of the unit cells are considered, but also different phase sensitivities are also required. Due to different frequency sensitivities of unit cells, two units with the same phase response at the initial frequency may have different phase responses at higher frequency. To describe the frequency coding property of unit cell, digitalized frequency sensitivity is proposed, in which the units are encoded with digits "0" and "1" to represent the low and high phase sensitivities, respectively. By this merit, two degrees of freedom, spatial coding and frequency coding, are obtained to control the EM energy radiations by a new class of frequency-spatial coding metamaterials. The above concepts and physical phenomena are confirmed by numerical simulations and experiments.

THE NISSL SUBSTANCE OF LIVING AND FIXED SPINAL GANGLION CELLS

PubMed Central

Deitch, Arline D.; Moses, Montrose J.

1957-01-01

Living chick spinal ganglion neurons grown for 19 to 25 days in vitro were photographed with a color-translating ultraviolet microscope (UV-91) at 265, 287, and 310 mµ. This instrument was unique in permitting rapid accumulation of ultraviolet information with minimal damage to the cell. In the photographs taken at 265 mµ of the living neurons, discrete ultraviolet-absorbing cytoplasmic masses were observed which were found to be virtually unchanged in appearance after formalin fixation. These were identical with the Nissl bodies of the same cells seen after staining with basic dyes. The correlation of ultraviolet absorption, ribonuclease extraction, and staining experiments with acid and basic dyes confirmed the ribonucleoprotein nature of these Nissl bodies in the living and fixed cells. No change in distribution or concentration of ultraviolet-absorbing substance was observed in the first 12 ultraviolet photographs of a neuron, and it is concluded that the cells had not been subjected to significant ultraviolet damage during the period of photography. On the basis of these observations, as well as previous findings with phase contrast microscopy, it is concluded that Nissl bodies preexist in the living neuron as discrete aggregates containing high concentrations of nucleoprotein. PMID:13438929

Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

PubMed

de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

2010-12-15

Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

In situ hybridisation of EBV DNA-DNA hybrids using wet heat in polypropylene containers.

PubMed Central

Labrecque, L G

1992-01-01

AIMS: To explore procedures designed to optimise DNA-DNA in situ hybridisation, using cells infected with Epstein-Barr virus (EBV) and tissues and subfragments of the EBV DNA as probes. METHODS: The denaturation step occurred in a polypropylene container, using wet heat generated by a hot water container, the pressure cooker, or the microwave oven, without coverslips, reaching a temperature of 121 degrees C or more in these two last systems. Two different visualisation systems were used. RESULTS: Fixed cells and tumours harbouring a high and medium to low copy number (a few hundreds to 33 copies per cell), were clearly labelled, using a simple reiterated subfragment (BamW) of the EBV DNA, and fresh frozen cells, harbouring a very low copy number (one to two on average) labelled using BamW as well as BamH (single non-reiterated 6 kilobase subfragment). CONCLUSION: This is a valuable alternative technique for DNA-DNA ISH that can be used in fresh frozen samples as well as fixed samples. Images PMID:1336018

A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

PubMed Central

Komatsu, Tetsuro; Dacheux, Denis; Kreppel, Florian; Nagata, Kyosuke; Wodrich, Harald

2015-01-01

Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes. PMID:26332038

Lower percentage of CD8+ T cells in peripheral blood of patients with sporotrichosis.

PubMed

Zhu, Mingji; Xu, Yaqin; An, Lin; Jiang, Jinlan; Zhang, Xu; Jiang, Rihua

2016-07-01

To characterize the peripheral immunity and immunity response of patients with sporotrichosis, in this study we determined the lymphocyte subsets in the peripheral blood of Chinese patients with sporotrichosis. In this retrospective study, peripheral blood was collected from 69 sporotrichosis patients (37, fixed cutaneous form; 32 lymphocutaneous) and 66 healthy controls. Lymphocyte subsets were analyzed using flow cytometry. Compared to controls, the percentage of CD8+ T cells was lower in sporotrichosis patients. The percentage of CD8+ T cells in peripheral blood tended to become lower with disease duration and disease severity, although the difference was not statistically significant for either acute, subacute and chronic patients or fixed cutaneous and lymphocutaneous patients. Our data indicate that the decrease of CD8+ T cells in peripheral blood of patients with sporotrichosis is associated with disease severity, although the difference was not statistically significant for either duration or clinical forms of the disease. Combining antifungal agents and immunomodulators in patients with long disease duration and lymphocutaneous may be more beneficial than antifungal monotherapy. Copyright © 2016. Published by Elsevier Inc.

Representing perturbed dynamics in biological network models

NASA Astrophysics Data System (ADS)

Stoll, Gautier; Rougemont, Jacques; Naef, Felix

2007-07-01

We study the dynamics of gene activities in relatively small size biological networks (up to a few tens of nodes), e.g., the activities of cell-cycle proteins during the mitotic cell-cycle progression. Using the framework of deterministic discrete dynamical models, we characterize the dynamical modifications in response to structural perturbations in the network connectivities. In particular, we focus on how perturbations affect the set of fixed points and sizes of the basins of attraction. Our approach uses two analytical measures: the basin entropy H and the perturbation size Δ , a quantity that reflects the distance between the set of fixed points of the perturbed network and that of the unperturbed network. Applying our approach to the yeast-cell-cycle network introduced by Li [Proc. Natl. Acad. Sci. U.S.A. 101, 4781 (2004)] provides a low-dimensional and informative fingerprint of network behavior under large classes of perturbations. We identify interactions that are crucial for proper network function, and also pinpoint functionally redundant network connections. Selected perturbations exemplify the breadth of dynamical responses in this cell-cycle model.

Compact fixed wavelength femtosecond oscillators as an add-on for tunable Ti:sapphire lasers extend the range of applications towards multimodal imaging and optogenetics

NASA Astrophysics Data System (ADS)

Hakulinen, T.; Klein, J.

2016-03-01

Two-photon (2P) microscopy based on tunable Ti:sapphire lasers has become a widespread tool for 3D imaging with sub-cellular resolution in living tissues. In recent years multi-photon microscopy with simpler fixed-wavelength femtosecond oscillators using Yb-doped tungstenates as gain material has raised increasing interest in life-sciences, because these lasers offer one order of magnitude more average power than Ti:sapphire lasers in the wavelength range around 1040 nm: Two-photon (2P) excitation of mainly red or yellow fluorescent dyes and proteins (e.g. YFP, mFruit series) simultaneously has been proven with a single IR laser wavelength. A new approach is to extend the usability of existing tunable Titanium sapphire lasers by adding a fixed IR wavelength with an Yb femtosecond oscillator. By that means a multitude of applications for multimodal imaging and optogenetics can be supported. Furthermore fs Yb-lasers are available with a repetition rate of typically 10 MHz and an average power of typically 5 W resulting in pulse energy of typically 500 nJ, which is comparably high for fs-oscillators. This makes them an ideal tool for two-photon spinning disk laser scanning microscopy and holographic patterning for simultaneous photoactivation of large cell populations. With this work we demonstrate that economical, small-footprint Yb fixed-wavelength lasers can present an interesting add-on to tunable lasers that are commonly used in multiphoton microscopy. The Yb fs-lasers hereby offer higher power for imaging of red fluorescent dyes and proteins, are ideally enhancing existing Ti:sapphire lasers with more power in the IR, and are supporting pulse energy and power hungry applications such as spinning disk microscopy and holographic patterning.

Co-C and Pd-C Fixed Points for the Evaluation of Facilities and Scales Realization at INRIM and NMC

NASA Astrophysics Data System (ADS)

Battuello, M.; Wang, L.; Girard, F.; Ang, S. H.

2014-04-01

Two hybrid cells for realizing the Co-C and Pd-C fixed points and constructed at Istituto Nazionale di Ricerca Metrologica (INRIM) were used for an evaluation of facilities and procedures adopted by INRIM and National Metrology Institute of Singapore (NMC) for the realization of the solid-liquid phase transitions of high-temperature fixed points and for determining their transition temperatures. Four different furnaces were used for the investigations, i.e., two single-zone furnaces, one of them of the direct-heating type, and two identical three-zone furnaces. The transition temperatures were measured at both institutes by adopting different procedures for realizing the radiation scales, i.e., at INRIM a scheme based on the extrapolation of fixed-point interpolated scales and an International Temperature Scale of 1990 (ITS-90) approach at NMC. The point of inflection (POI) of the melting curves was determined and assumed as a practical representation of the melting temperature. Different methods for deriving the POI were used, and differences as large as some hundredths of a kelvin were found with the different approaches. The POIs of the different melting curves were analyzed with respect to the different possible operative conditions with the aim of deriving reproducibility figures to improve the estimated uncertainty. As regard to the institutes inter-comparison, differences of 0.13 K and 0.29 K were found between INRIM and NMC determinations at the Co-C and Pd-C points, respectively. Such differences are compatible with the combined standard uncertainties of the comparison, which are estimated to be 0.33 K and 0.36 K at the Co-C and Pd-C points, respectively.

Co-C and Pd-C Eutectic Fixed Points for Radiation Thermometry and Thermocouple Thermometry

NASA Astrophysics Data System (ADS)

Wang, L.

2017-12-01

Two Co-C and Pd-C eutectic fixed point cells for both radiation thermometry and thermocouple thermometry were constructed at NMC. This paper describes details of the cell design, materials used, and fabrication of the cells. The melting curves of the Co-C and Pd-C cells were measured with a reference radiation thermometer realized in both a single-zone furnace and a three-zone furnace in order to investigate furnace effect. The transition temperatures in terms of ITS-90 were determined to be 1324.18 {°}C and 1491.61 {°}C with the corresponding combined standard uncertainty of 0.44 {°}C and 0.31 {°}C for Co-C and Pd-C, respectively, taking into account of the differences of two different types of furnaces used. The determined ITS-90 temperatures are also compared with that of INRIM cells obtained using the same reference radiation thermometer and the same furnaces with the same settings during a previous bilateral comparison exercise (Battuello et al. in Int J Thermophys 35:535-546, 2014). The agreements are within k=1 uncertainty for Co-C cell and k = 2 uncertainty for Pd-C cell. Shapes of the plateaus of NMC cells and INRIM cells are compared too and furnace effects are analyzed as well. The melting curves of the Co-C and Pd-C cells realized in the single-zone furnace are also measured by a Pt/Pd thermocouple, and the preliminary results are presented as well.

Evolutionary dynamics of adult stem cells: comparison of random and immortal-strand segregation mechanisms.

PubMed

Tannenbaum, Emmanuel; Sherley, James L; Shakhnovich, Eugene I

2005-04-01

This paper develops a point-mutation model describing the evolutionary dynamics of a population of adult stem cells. Such a model may prove useful for quantitative studies of tissue aging and the emergence of cancer. We consider two modes of chromosome segregation: (1) random segregation, where the daughter chromosomes of a given parent chromosome segregate randomly into the stem cell and its differentiating sister cell and (2) "immortal DNA strand" co-segregation, for which the stem cell retains the daughter chromosomes with the oldest parent strands. Immortal strand co-segregation is a mechanism, originally proposed by [Cairns Nature (London) 255, 197 (1975)], by which stem cells preserve the integrity of their genomes. For random segregation, we develop an ordered strand pair formulation of the dynamics, analogous to the ordered strand pair formalism developed for quasispecies dynamics involving semiconservative replication with imperfect lesion repair (in this context, lesion repair is taken to mean repair of postreplication base-pair mismatches). Interestingly, a similar formulation is possible with immortal strand co-segregation, despite the fact that this segregation mechanism is age dependent. From our model we are able to mathematically show that, when lesion repair is imperfect, then immortal strand co-segregation leads to better preservation of the stem cell lineage than random chromosome segregation. Furthermore, our model allows us to estimate the optimal lesion repair efficiency for preserving an adult stem cell population for a given period of time. For human stem cells, we obtain that mispaired bases still present after replication and cell division should be left untouched, to avoid potentially fixing a mutation in both DNA strands.

Evolutionary dynamics of adult stem cells: Comparison of random and immortal-strand segregation mechanisms

NASA Astrophysics Data System (ADS)

Tannenbaum, Emmanuel; Sherley, James L.; Shakhnovich, Eugene I.

2005-04-01

This paper develops a point-mutation model describing the evolutionary dynamics of a population of adult stem cells. Such a model may prove useful for quantitative studies of tissue aging and the emergence of cancer. We consider two modes of chromosome segregation: (1) random segregation, where the daughter chromosomes of a given parent chromosome segregate randomly into the stem cell and its differentiating sister cell and (2) “immortal DNA strand” co-segregation, for which the stem cell retains the daughter chromosomes with the oldest parent strands. Immortal strand co-segregation is a mechanism, originally proposed by [Cairns Nature (London) 255, 197 (1975)], by which stem cells preserve the integrity of their genomes. For random segregation, we develop an ordered strand pair formulation of the dynamics, analogous to the ordered strand pair formalism developed for quasispecies dynamics involving semiconservative replication with imperfect lesion repair (in this context, lesion repair is taken to mean repair of postreplication base-pair mismatches). Interestingly, a similar formulation is possible with immortal strand co-segregation, despite the fact that this segregation mechanism is age dependent. From our model we are able to mathematically show that, when lesion repair is imperfect, then immortal strand co-segregation leads to better preservation of the stem cell lineage than random chromosome segregation. Furthermore, our model allows us to estimate the optimal lesion repair efficiency for preserving an adult stem cell population for a given period of time. For human stem cells, we obtain that mispaired bases still present after replication and cell division should be left untouched, to avoid potentially fixing a mutation in both DNA strands.

Trapped charge densities in Al{sub 2}O{sub 3}-based silicon surface passivation layers

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

Jordan, Paul M., E-mail: Paul.Jordan@namlab.com; Simon, Daniel K.; Dirnstorfer, Ingo

2016-06-07

In Al{sub 2}O{sub 3}-based passivation layers, the formation of fixed charges and trap sites can be strongly influenced by small modifications in the stack layout. Fixed and trapped charge densities are characterized with capacitance voltage profiling and trap spectroscopy by charge injection and sensing, respectively. Al{sub 2}O{sub 3} layers are grown by atomic layer deposition with very thin (∼1 nm) SiO{sub 2} or HfO{sub 2} interlayers or interface layers. In SiO{sub 2}/Al{sub 2}O{sub 3} and HfO{sub 2}/Al{sub 2}O{sub 3} stacks, both fixed charges and trap sites are reduced by at least a factor of 5 compared with the value measured inmore » pure Al{sub 2}O{sub 3}. In Al{sub 2}O{sub 3}/SiO{sub 2}/Al{sub 2}O{sub 3} or Al{sub 2}O{sub 3}/HfO{sub 2}/Al{sub 2}O{sub 3} stacks, very high total charge densities of up to 9 × 10{sup 12} cm{sup −2} are achieved. These charge densities are described as functions of electrical stress voltage, time, and the Al{sub 2}O{sub 3} layer thickness between silicon and the HfO{sub 2} or the SiO{sub 2} interlayer. Despite the strong variation of trap sites, all stacks reach very good effective carrier lifetimes of up to 8 and 20 ms on p- and n-type silicon substrates, respectively. Controlling the trap sites in Al{sub 2}O{sub 3} layers opens the possibility to engineer the field-effect passivation in the solar cells.« less

Quantifying the contribution of single microbial cells to nitrogen assimilation in aquatic environments

NASA Astrophysics Data System (ADS)

Musat, N.; Kuypers, M. M. M.

2009-04-01

Nitrogen is a primary productivity-limiting nutrient in the ocean. The nitrogen limitation of productivity may be overcome by organisms capable of converting dissolved N2 into fixed nitrogen available to the ecosystem. In many oceanic regions, growth of phytoplankton is nitrogen limited because fixation of N2 cannot make up for the removal of fixed inorganic nitrogen (NH4+, NO2-, NO3-) by anaerobic microbial processes. The amount of available fixed nitrogen in the ocean can be changed by the biological processes of heterotrophic denitrification, anaerobic ammonium oxidation and nitrogen fixation. For a complete understanding of nitrogen cycling in the ocean a link between the microbial and biogeochemical processes at the single cell level and their role in global biogeochemical cycles is essential. Here we report a recently developed method, Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS) and its potential application to study the nitrogen-cycle processes in the ocean. The method allows simultaneous phylogenetic identification and quantitation of metabolic activities of single microbial cells in the environment. It uses horseradish-peroxidase-labeled oligonucleotide probes and fluorine-containing tyramides for the identification of microorganisms in combination with stable-isotope-labeling experiments for analyzing the metabolic function of single microbial cells. HISH-SIMS was successfully used to study nitrogen assimilation and nitrogen fixation by anaerobic phototrophs in a meromictic alpine lake. The HISH-SIMS method enables studies of the ecophysiology of individual, phylogenetically identified microorganisms involved in the N-cycle and allows us to track the flow of nitrogen within microbial communities.

Localization-based super-resolution imaging meets high-content screening.

PubMed

Beghin, Anne; Kechkar, Adel; Butler, Corey; Levet, Florian; Cabillic, Marine; Rossier, Olivier; Giannone, Gregory; Galland, Rémi; Choquet, Daniel; Sibarita, Jean-Baptiste

2017-12-01

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

Connect-disconnect coupling for preadjusted rigid shafts

NASA Technical Reports Server (NTRS)

Bajkowski, F. W.; Holmberg, A.

1969-01-01

Coupling device enables a rigid shaft to be connected to or disconnected from a fixed base without disturbing the point of adjustment of the shaft in a socket or causing the shaft to rotate. The coupling consists of an externally threaded, internally slotted boss extending from the fixed base.

Fibroblast Activation Protein-Alpha, a Serine Protease that Facilitates Metastasis by Modification of Diverse Microenvironments

DTIC Science & Technology

2011-10-01

lung tissue. We were not able to detect sufficient numbers of cells in this manner. We tried a different procedure for fixing the lungs after they...added after 24 hours. The films were fixed and evaluated microscopically. In four trials, 10 random microscopic fields were selected and... dosing by oral gavage once daily with 1.3 mg/kg L-valine-L-boroproline called talabostat (extracellular & intracellular DASH), 13.3 mg/kg L- glutamyl

Application of GFP technique for cytoskeleton visualization onboard the International Space Station.

PubMed

Kordyum, E L; Shevchenko, G V; Yemets, A I; Nyporko, A I; Blume, Ya B

2005-03-01

Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensing and trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different modifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles, they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocytochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletal visualization onboard the ISS, namely, application of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. The creation of chimerical protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of the cytoskeleton functional status in microgravity. c2004 Elsevier Ltd. All rights reserved.