Reconstituted human gingival epithelium: nonsubmerged in vitro model.

PubMed

Delcourt-Huard, A; Corlu, A; Joffre, A; Magloire, H; Bonnaure-Mallet, M

1997-01-01

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

PubMed

Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

2013-12-01

Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

PubMed

Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

2009-08-01

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

Enamel tissue engineering using subcultured enamel organ epithelial cells in combination with dental pulp cells.

PubMed

Honda, Masaki J; Shinmura, Yuka; Shinohara, Yoshinori

2009-01-01

We describe a strategy for the in vitro engineering of enamel tissue using a novel technique for culturing enamel organ epithelial (EOE) cells isolated from the enamel organ using 3T3-J2 cells as a feeder layer. These subcultured EOE cells retain the capacity to produce enamel structures over a period of extended culture. In brief, enamel organs from 6-month-old porcine third molars were dissociated into single cells and subcultured on 3T3-J2 feeder cell layers. These subcultured EOE cells were then seeded onto a collagen sponge in combination with primary dental pulp cells isolated at an early stage of crown formation, and these constructs were transplanted into athymic rats. After 4 weeks, complex enamel-dentin structures were detected in the implants. These results show that our culture technique maintained ameloblast lineage cells that were able to produce enamel in vivo. This novel subculture technique provides an important tool for tooth tissue engineering. Copyright 2008 S. Karger AG, Basel.

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation.

PubMed

Behr, Rüdiger; Heneweer, Carola; Viebahn, Christoph; Denker, Hans-Werner; Thie, Michael

2005-01-01

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Growth factor expression pattern of homologous feeder layer for culturing buffalo embryonic stem cell-like cells.

PubMed

Sharma, Ruchi; George, Aman; Kamble, Nitin M; Chauhan, Manmohan S; Singla, Suresh; Manik, Radhey S; Palta, Prabhat

2012-01-01

The present study examined the expression profile of buffalo fetal fibroblasts (BFF) used as a feeder layer for embryonic stem (ES) cell-like cells. The expression of important growth factors was detected in cells at different passages. Mitomycin-C inactivation increased relative expression levels of ACTIVIN-A, TGF-β1, BMP-4 and GREMLIN but not of fibroblast growth factor-2 (FGF-2). The expression level of ACTIVIN-A, transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-4 (BMP-4) and FGF-2 was similar in buffalo fetal fibroblast (BFF) cultured in stem cell medium (SCM), SCM+1000IU mL(-1) leukemia inhibitory factor (LIF), SCM+5 ngmL(-1) FGF-2 or SCM+LIF+FGF-2 for 24 h whereas GREMLIN expression was higher in FGF-2-supplemented groups. In spent medium, the concentration of ACTIVIN-A was higher in FGF-2-supplemented groups whereas that of TGF-β1 was similar in SCM and LIF+FGF-2, which was higher than when either LIF or FGF-2 was used alone. Following culture of ES cell-like cells on a feeder layer for 24 h, the TGF-β1 concentration was higher with LIF+FGF-2 than with LIF or FGF-2 alone which, in turn, was higher than that in SCM. In the LIF+FGF-2 group, the concentration of TGF-β1 was lower and that of ACTIVIN-A was higher in spent medium at 24 h than at 48 h of culture. These results suggest that BFF produce signalling molecules that may help in self-renewal of buffalo ES cell-like cells.

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

PubMed Central

Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

2013-01-01

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

PubMed

Gómez, M C; Serrano, M A; Pope, C Earle; Jenkins, J A; Biancardi, M N; López, M; Dumas, C; Galiguis, J; Dresser, B L

2010-09-01

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines. Copyright 2010 Elsevier Inc. All rights reserved.

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

PubMed Central

Garitaonandia, Ibon; Amir, Hadar; Boscolo, Francesca Sesillo; Wambua, Gerald K.; Schultheisz, Heather L.; Sabatini, Karen; Morey, Robert; Waltz, Shannon; Wang, Yu-Chieh; Tran, Ha; Leonardo, Trevor R.; Nazor, Kristopher; Slavin, Ileana; Lynch, Candace; Li, Yingchun; Coleman, Ronald; Gallego Romero, Irene; Altun, Gulsah; Reynolds, David; Dalton, Stephen; Parast, Mana; Loring, Jeanne F.; Laurent, Louise C.

2015-01-01

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies. PMID:25714340

Substrate stiffness affects skeletal myoblast differentiation in vitro

NASA Astrophysics Data System (ADS)

Romanazzo, Sara; Forte, Giancarlo; Ebara, Mitsuhiro; Uto, Koichiro; Pagliari, Stefania; Aoyagi, Takao; Traversa, Enrico; Taniguchi, Akiyoshi

2012-12-01

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ɛ-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions☆

PubMed Central

Soares, Filipa A.C.; Chandra, Amit; Thomas, Robert J.; Pedersen, Roger A.; Vallier, Ludovic; Williams, David J.

2014-01-01

The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes. PMID:24440272

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

PubMed

Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

2011-11-15

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions.

PubMed

McLane, J A; Katz, M; Abdelkader, N

1990-04-01

1,25-Dihydroxyvitamin D3 (1,25-(OH)2-D3) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)2-D3 using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)2-D3. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)2-D3. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)2-D3. The antiproliferative effect of 1,25-(OH)2-D3 was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)2-D3 is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)2-D3 to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)2-D3, but can be used to assay other agents that modulate keratinocyte proliferation.

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

PubMed

Roy, A; Krzykwa, E; Lemieux, R; Néron, S

2001-12-01

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

PubMed

Kaneko, Ai; Sankai, Yoshiyuki

2014-01-01

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

The effect of isolation and culture methods on epithelial stem cell populations and their progeny-toward an improved cell expansion protocol for clinical application.

PubMed

Lenihan, Catherine; Rogers, Caroline; Metcalfe, Anthony D; Martin, Yella H

2014-12-01

The use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells. We compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin. During the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower. Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders.

PubMed

Teotia, Pooja; Sharma, Shilpa; Airan, Balram; Mohanty, Sujata

2016-12-01

Human embryonic stem cell (hESC) lines are commonly maintained on inactivated feeder cells, in the medium supplemented with basic fibroblast growth factor (bFGF). However, limited availability of feeder cells in culture, and the high cost of growth factors limit their use in scalable expansion of hESC cultures for clinical application. Here, we describe an efficient and cost-effective feeder and bFGF-free culture of hESCs using conditioned medium (CM) from immortalized feeder cells. KIND-1 hESC cell line was cultured in CM, collected from primary mouse embryonic fibroblast, human foreskin fibroblast (HFF) and immortalized HFF (I-HFF). Pluripotency of KIND-1 hESC cell line was confirmed by expression of genes, proteins and cell surface markers. In culture, these cells retained normal morphology, expressed all cell surface markers, could differentiate to embryoid bodies upon culture in vitro. Furthermore, I-HFF feeder cells without supplementation of bFGF released ample amount of endogenous bFGF to maintain stemness of hESC cells. The study results described the use of CM from immortalized feeder cells as a consistent source and an efficient, inexpensive feeder-free culture system for the maintenance of hESCs. Moreover, it was possible to maintain hESCs without exogenous supplementation of bFGF. Thus, the study could be extended to scalable expansion of hESC cultures for therapeutic purposes.

Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

PubMed

Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

2009-11-01

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

PubMed

Behboodi, E; Bondareva, A; Begin, I; Rao, K; Neveu, N; Pierson, J T; Wylie, C; Piero, F D; Huang, Y J; Zeng, W; Tanco, V; Baldassarre, H; Karatzas, C N; Dobrinski, I

2011-03-01

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells. Copyright © 2011 Wiley-Liss, Inc.

Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three-dimensional multilayered patty-like spheroids in feeder-free culture and maintain their pluripotency.

PubMed

Alamein, Mohammad A; Wolvetang, Ernst J; Ovchinnikov, Dmitry A; Stephens, Sebastien; Sanders, Katherine; Warnke, Patrick H

2015-09-01

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. Copyright © 2014 John Wiley & Sons, Ltd.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

2010-01-01

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

PubMed

Nakajima, Ryota; Takeda, Shizu

2014-01-01

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

Sakai, Yusuke; Koike, Makiko; Hasegawa, Hideko; Yamanouchi, Kosho; Soyama, Akihiko; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Ohashi, Kazuo; Okano, Teruo; Eguchi, Susumu

2013-01-01

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

Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

PubMed

Maruyama, C L M; Leigh, N J; Nelson, J W; McCall, A D; Mellas, R E; Lei, P; Andreadis, S T; Baker, O J

2015-11-01

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features. © International & American Associations for Dental Research 2015.

Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

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

Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu

2014-10-24

Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice.more » Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.« less

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting.

PubMed

Wong, Chui-Wei; Chen, You-Tzung; Chien, Chung-Liang; Yu, Tien-Yu; Rwei, Syang-Peng; Hsu, Shan-Hui

2018-01-01

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

PubMed Central

Sakai, Yusuke; Koike, Makiko; Hasegawa, Hideko; Yamanouchi, Kosho; Soyama, Akihiko; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Ohashi, Kazuo; Okano, Teruo; Eguchi, Susumu

2013-01-01

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions. PMID:23923035

Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

PubMed Central

Zhao, Zhenqiang; Ma, Yanlin; Chen, Zhibin; Liu, Qian; Li, Qi; Kong, Deyan; Yuan, Kunxiong; Hu, Lan; Wang, Tan; Chen, Xiaowu; Peng, Yanan; Jiang, Weimin; Yu, Yanhong; Liu, Xinfeng

2016-01-01

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons. PMID:28066186

Atmospheric Characteristics of Cool Season Intermittent Precipitation Near Portland, Oregon

NASA Astrophysics Data System (ADS)

Cunningham, Jeffrey Glenn

Pacific Northwest cool season precipitation is often described as mostly stratiform (i.e. steady and continuous). While most regional precipitation is stratiform in terms of area and duration, embedded convective cells within stratiform precipitation occur frequently enough to warrant study. Embedded cells locally increase rain rate, total precipitation, and streamflow discharge and hence raise the risk of flooding, landslides, and debris flows. Analysis of vertically pointing radar data near Portland, Oregon for three cool seasons (2005 to 2008) indicates that fallstreaks in the snow layer, locally enhanced precipitation regions a few kilometers in size indicated in radar reflectivity data above the 0° C altitude, are nearly ubiquitous on days with significant rainfall accumulation and large areas of precipitation. The observed fallstreaks in snow enhance rainfall immediately below the snow fallstreak. Compared to stratiform periods, embedded convective periods include higher Doppler vertical velocity values and higher variability in velocities especially in the snow layer. The combination of these findings points to generating cells within the snow layer and the seeder-feeder mechanism as important sources of surface precipitation variability for periods of embedded convective cells within stratiform precipitation. The primary goal of this study was to determine the sources of instability typically associated with convective cells embedded within stratiform precipitation for Pacific Northwest cool season storms. Storm periods occurring over six cool seasons (2002 to 2008, totaling 1923 hours) of operational radar data (KRTX) and 166 upper air soundings (KSLE) are analyzed. A new method was employed to objectively determine the degree of precipitation intermittency in sequences of radar scans. The resulting continuum of intermittency values was grouped into four categories: mostly convective precipitation, mostly stratiform precipitation, embedded convective cells within stratiform precipitation, and other. Atmospheric soundings during periods with embedded convective cells within stratiform precipitation are more likely to have convective available potential energy (CAPE) than soundings during periods of mostly stratiform precipitation. Specifically, most unstable parcel CAPE (MUCAPE) > 0 J kg-1 occurs 2.8 more frequently during embedded periods than for mostly stratiform periods. Over 90% of embedded periods have MUCAPE > 0 J kg-1 or at least two 500 meter layers of potential instability. In contrast to the near surface based instability most commonly associated with the mostly convective precipitation, embedded convection is elevated. The median most unstable parcel height of origin for embedded convective periods is 2.5 km compared to 0.5 km for mostly convective periods. Although this present research did not deal directly with orographic precipitation enhancement, it does address synoptic and mesoscale precipitation processes that frequently occur near terrain in the Pacific Northwest. The exclusion of the seeder-feeder mechanism as a mode of cellularity for orographic precipitation in recent work is inconsistent with the observations presented here and inconsistent with much of the pre-2000 literature, which show the seeder-feeder mechanism directly modulating surface rain rate with or without terrain present. Numerical models, whether operational or idealized, need to represent the seeder-feeder process in order to accurately simulate precipitation variability at small spatial scales (less than < 5-10 km) and temporal scales (< 3 hours) within the warm sector of Pacific Northwest extratropical cyclones.

An evaluation of the choice of feeder cell growth arrest for the production of cultured epidermis.

PubMed

Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

2015-12-01

Growth arrested 3T3 cells have been used as feeder cells in human epidermal keratinocyte cultures to produce cultured epidermal autografts for the treatment of burns. The feeder cells were ideally growth-arrested by gamma-irradiation. Alternatively, growth arrest by mitomycin C treatment is a cost effective option. We compared the functional efficacy of these two approaches in keratinocyte cultures by colony forming efficiency, the net growth area of colonies, BrdU labeling and histological features of cultured epidermal sheets. The growth area estimation involved a semi-automated digital technique using the Adobe Photoshop and comprised of isolation and enumeration of red pixels in Rhodamine B-stained keratinocyte colonies. A further refinement of the technique led to the identification of critical steps to increasing the degree of accuracy and enabling its application as an extension of colony formation assay. The results on feeder cell functionality revealed that the gamma irradiated feeders influenced significantly higher colony forming efficiency and larger growth area than the mitomycin C treated feeders. The BrdU labeling study indicated significant stimulation of the overall keratinocyte proliferation by the gamma irradiated feeders. The cultured epidermal sheets produced by gamma feeders were relatively thicker than those produced by mitomycin C feeders. We discussed the clinical utility of mitomycin C feeders from the viewpoint of cost-effective burn care in developing countries. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

PubMed

Yu, G; Okawa, H; Okita, K; Kamano, Y; Wang, F; Saeki, M; Yatani, H; Egusa, H

2016-01-01

Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs. © International & American Associations for Dental Research 2015.

Diatom feeding across trophic guilds in tidal flat nematodes, and the importance of diatom cell size

NASA Astrophysics Data System (ADS)

Moens, Tom; Vafeiadou, Anna-Maria; De Geyter, Ellen; Vanormelingen, Pieter; Sabbe, Koen; De Troch, Marleen

2014-09-01

We examine the capacity of nematodes from three feeding types (deposit feeder, epistrate feeder, predator) to utilize microphytobenthos (MPB), and assess whether diatom cell size and consumer body size are important drivers of their feeding. We analyzed natural stable isotope ratios of carbon and nitrogen in abundant nematode genera and a variety of carbon sources at an estuarine intertidal flat. All nematodes had δ13C indicating that MPB is their major carbon source. δ15N, however, demonstrated that only one deposit and one epistrate feeder genus obtained most of their carbon from direct grazing on MPB, whereas other deposit feeders and predators obtained at least part of their carbon by predation on MPB grazers. We then performed a microcosm experiment in which equal cell numbers of each of three differently sized strains of the pennate diatom Seminavis were offered as food to four, one and one genera of deposit feeders, epistrate feeders and predators, respectively. Previous studies have shown that all but the epistrate feeder ingest whole diatoms, whereas the epistrate feeder pierces cells and sucks out their contents. Most genera showed markedly higher carbon absorption from medium and large cells than from small ones. When considering the number of cells consumed, however, none of the nematodes which ingest whole cells exhibited a clear preference for any specific diatom size. The epistrate feeder was the smallest nematode taxon considered here, yet it showed a marked preference for large cells. These results highlight that the feeding mechanism is much more important than consumer size as a driver of particle size selection in nematodes grazing MPB.

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

PubMed Central

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

Postmitotic human dermal fibroblasts preserve intact feeder properties for epithelial cell growth after long-term cryopreservation.

PubMed

Limat, A; Hunziker, T; Boillat, C; Noser, F; Wiesmann, U

1990-07-01

In vitro, human dermal fibroblasts (HDF) differentiate through morphologically and biochemically identified compartments. In the course of this spontaneous differentiation through mitotic and postmitotic states, a tremendous increase in cellular and nuclear size occurs. Induction of postmitotic states can be accelerated by chemical (e.g., mitomycin C) or physical (e.g., x-ray) treatments. Such experimentally induced postmitotic HDF cells support very efficiently the growth of cutaneous epithelial cells, i.e. interfollicular keratinocytes and follicular outer root sheath cells, especially in primary cultures starting from very low cell seeding densities. The HDF feeder system provides more fundamental and also practical advantages, i.e. use of initially diploid human fibroblasts from known anatomic locations, easy handling and excellent reproducibility, and the possibility of long-term storage by incubation at 37 degrees C. Conditions for the cryogenic storage of postmitotic HDF cells in liquid nitrogen are presented and related to the feeder capacity for epithelial cell growth. Because postmitotic HDF cells preserve intact feeder properties after long-term storage, the immediate availability of feeder cells and the possibility to repeat experiments with identical materials further substantiate the usefulness of this feeder system.

Exposure cell number during feeder cell growth-arrest by Mitomycin C is a critical pharmacological aspect in stem cell culture system.

PubMed

Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

2016-01-01

Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner. Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number. The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10μg/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure. The in vitro cell density titration can facilitate prediction of a compound's operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of cultivated murine lacrimal gland epithelial cells

PubMed Central

Kobayashi, Shinya; Kawashima, Motoko; Okada, Naoko; Mishima, Kenji; Saito, Ichiro; Ito, Masataka; Shimmura, Shigeto; Tsubota, Kazuo

2012-01-01

Purpose To date, mouse lacrimal gland epithelial cells have been cultured successfully but only in cases involving newborn mouse lacrimal glands. In this work, we attempted to cultivate and characterize adult mouse lacrimal gland epithelial cells. Methods Lacrimal glands were removed from newborn mice (C57B/6) and isolated lacrimal gland epithelial cells were seeded onto tissue culture treated or low adherent culture dishes in Cnt-07 culture medium with or without cholera toxin. Cultivated cells were characterized by immunostaining with pan-cytokeratin, α-smooth muscle actin, and lactoferrin antibodies. Lacrimal gland cells from 7-week-old green fluorescent protein (GFP) and non-GFP (C57B/6) mice were mixed and seeded onto uncoated dishes to assess sphere-forming efficiency. Cells were also seeded onto 3T3 cell feeder layers to assess colony forming efficiency. Results Lacrimal gland epithelial cells were selectively cultured with cholera toxin, and cell type was verified by pan-cytokeratin and α-smooth muscle actin immunostaining. Sphere formation from single cells of adult mice was observed using specific medium and low adherent culture dishes. These cells could also undergo colony formation on 3T3 feeder cells. Conclusions Adult mouse lacrimal gland epithelial cells were successfully cultivated in cholera toxin-containing medium, and were observed to form spheres from single cells. PMID:22665974

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

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

Singla, Dinender K.; Schneider, David J.; LeWinter, Martin M.

2006-06-30

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not.more » Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.« less

Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

NASA Astrophysics Data System (ADS)

Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

2016-02-01

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

Process-Based Expansion and Neural Differentiation of Human Pluripotent Stem Cells for Transplantation and Disease Modeling

PubMed Central

Stover, Alexander E.; Brick, David J.; Nethercott, Hubert E.; Banuelos, Maria G.; Sun, Lei; O’Dowd, Diane K.; Schwartz, Philip H.

2014-01-01

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials. PMID:23893392

An in vitro monocyte culture method and establishment of a human monocytic cell line (K63).

PubMed

Kadoi, Katsuyuki

2011-01-01

A novel method of monocyte culture in vitro was developed. The fraction of monocytes was obtained by density centrifugation of heparinised human venous blood samples. Monocytes were suspended in a modified Rosewell Park Memorial Institute medium (RPMI)-1640 (mRPMI) supplemented with 10% non-inactivated autologous serum added to the feeder cells. An avian cell line was used for feeder cells. Only those monocytes that settled on feeder cells grew rapidly at 37°C-38°C into a formation of clumped masses within two to three days. The cell mass was harvested and subcultures were made without feeder cells. A stable cell line (K63) was established from subcultures using a limited dilution method and cell cloning in microplates. K63 cells were adapted for later growth in the mRPMI medium supplemented with 10% foetal calf serum. The cells were well maintained at over 50th passage levels. This method proved to be applicable for monocyte cultures of animals as well.

Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

PubMed

Kubota, Hiroshi; Wu, Xin; Goodyear, Shaun M; Avarbock, Mary R; Brinster, Ralph L

2011-08-01

Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

USDA-ARS?s Scientific Manuscript database

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

PubMed

Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

2018-01-22

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

PubMed

Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

2010-04-01

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line.

PubMed

Morita, Maresuke; Fujita, Naoki; Abe, Momoko; Hayashimoto, Koji; Nakagawa, Takayuki; Nishimura, Ryohei; Tsuzuki, Keiko

2018-06-01

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.

PubMed

Makoolati, Zohreh; Movahedin, Mansoureh; Forouzandeh-Moghadam, Mehdi

2016-12-01

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs. © 2016 The Author(s).

Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes.

PubMed

Limat, A; Hunziker, T; Boillat, C; Bayreuther, K; Noser, F

1989-05-01

For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

PubMed

Szaraz, Peter; Gratch, Yarden S; Iqbal, Farwah; Librach, Clifford L

2017-08-09

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

Quantitative and semi-quantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

USDA-ARS?s Scientific Manuscript database

Feeder-cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semi-quantitative immunoassays were performed...

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

PubMed

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts

PubMed Central

Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming; Wang, Yujiong; He, Yulong

2017-01-01

Induced pluripotent stem cells (iPS) represent an important tool to develop disease-modeling assays, drug testing assays and cell-based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), Kruppel-like factor 4 and c-Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder-free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin-coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases. PMID:28393187

Vertical linear feeder to elliptical igneous saucer-shaped sills: evidences from structural observations, geochemistry and experimental modeling

NASA Astrophysics Data System (ADS)

Galerne, C. Y.; Galland, O.; Neumann, E. R.; Planke, S.

2009-12-01

The structural relationships between sills and their feeders are poorly documented because they are rarely observed in the field and difficult to image on seismic data. For instance, it is unclear whether sills are fed by pipes, dikes or other sills. Nevertheless, the geometrical relationships between sills and their feeders provide first-order constraints on magma emplacement mechanisms. Here, we investigate the structural and geochemical relationships between sills and potential feeder dikes in a remarkably well-preserved and exposed sill complex, the Golden Valley Sill Complex (GVSC), Karoo Basin, South Africa. The GVSC consists of five major saucer-shaped sills and six dikes. The Golden Valley sill itself is an elliptical saucer, with a N-S trend. A one meter thick dike (D4) crops out underneath the southern tip of the Golden Valley sill. The strike of this dike is parallel to the long axis of the Golden Valley sill. Detailed sampling and geochemical analyses of the GVSC show that each sill and dike exhibits a specific geochemical signature. The Golden Valley sill and its underlying dike D4 have identical signatures. Although there is no clear structural evidence, the consistent geometrical and geochemical relationships between the Golden Valley sill and the D4 dike suggest that this vertical linear structure is the feeder of the overlying saucer-shaped sill. In order to investigate the relationships between sills and feeders, we resorted to scaled laboratory experiments. The experiments consisted of a low-viscosity vegetable oil representing magma and a cohesive fine-grained silica flour representing brittle rocks. We placed a horizontal weak layer into the silica flour, just above the top of the inlet, to simulate strata. Such a weak layer controlled the formation of horizontal sill that subsequently turned into a transgressive sheet leading to the formation of a saucer geometry. We ran experiments with varying inlet shapes: 1) a point inlet representing a pipe-like feeder and 2) a linear feeder representing a dike-like feeder. In the experiments with point inlet, circular saucer-shaped sills formed. In the experiments with linear feeder, elliptical saucer-shaped sills formed. In the latter experiments, the long axes of the saucers were parallel to, and located directly above, the linear feeder. The experiments show that the feeder geometry has an important influence on the geometry of the emplaced sills. There are close similarities between the geometry of the Golden Valley sill and the intrusions formed in the experiments. The elliptical shape of the Golden Valley sill suggests that it was fed by an elongated feeder, probably the D4 dike. In general, our results show that the three-dimensional geometry of saucer-shaped sills observed in sedimentary basins, may constrain the shape of their feeders, i.e. their emplacement mechanisms.

Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-β receptorII in co-culture with bone marrow mesenchymal stromal cells.

PubMed

Sohrabi Akhkand, Saman; Amirizadeh, Naser; Nikougoftar, Mahin; Alizadeh, Javad; Zaker, Farhad; Sarveazad, Arash; Joghataei, Mohammad Taghi; Faramarzi, Mahmood

2016-08-01

Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-β signaling pathway through reduction of TGF-βRII expression. CD34(+) cells were isolated from UCB and transfected by SiRNA targeting TGF-βRII mRNA. CD34(+) cells were expanded in four culture media with different conditions, including 1) expansion of CD34(+) cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-βRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-βRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture. The fold increase in CD34(+) cells, TNCs, and colony numbers (71.8±6.9, 93.2±10.2 and 128±10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0day) before expansion was statistically significant (P<0.05). The results showed that transfection of CD34(+) cells with SiRNA targeting TGF-βRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Construction of Artificial Hepatic Lobule-Like Spheroids on a Three-Dimensional Culture Device.

PubMed

Enosawa, Shin; Miyamoto, Yoshitaka; Kubota, Hisayo; Jomura, Tomoko; Ikeya, Takeshi

2012-01-01

One major purpose of cell culture is the reconstruction of physiological structures. Using bovine aortic epithelium cell line HH (JCRB0099) as feeder cells and rat primary hepatocytes, we constructed hepatic lobule-like spheroids on a cell array plate designed for three-dimensional (3D) culture. Microfabricated patterning of the cell array with poly(ethyleneglycol) brushes promotes the formation of spheroids at 100-μm diameter at 100-μm intervals. Our standard protocol is to seed with feeder HH cells and then seed with primary hepatic parenchymal cells. The composite cell spheroids thus obtained are called heterospheroids. Feeder cells that were attached to the plate migrated and encompassed the spheroidal hepatocyte mass. Electron microscopy revealed Disse space-like structures characterized by hepatocyte-rooted microvilli rooted between hepatocyte and feeder epithelial HH cells. Differentiated hepatic functions such as albumin synthesis and cytochrome P450 subfamily CYP3A activities were maintained for 28 days in the heterospheroid versus monospheroid and monolayer cultures. In addition, glucuronide conjugation activity was maintained at a high level in heterospheroids. These results indicate that structurally similar hepatic lobules were formed in a microfabricated cell array coculture system and that the culture conditions are beneficial for maintaining differentiated hepatic functions.

Activation and propagation of tumor infiltrating lymphocytes on clinical-grade designer artificial antigen presenting cells for adoptive immunotherapy of melanoma

PubMed Central

Forget, Marie-Andrée; Malu, Shruti; Liu, Hui; Toth, Christopher; Maiti, Sourindra; Kale, Charuta; Haymaker, Cara; Bernatchez, Chantale; Huls, Helen; Wang, Ena; Marincola, Francesco M.; Hwu, Patrick; Cooper, Laurence J. N.; Radvanyi, Laszlo G.

2014-01-01

PURPOSE Adoptive cell therapy (ACT) with autologous tumor infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as “feeders” to support a rapid expansion protocol (REP). Here, we optimized a platform to propagate TIL to a clinical scale using K562-cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand and membrane-bound IL-15 to function as artificial antigen-presenting cell (aAPC) as an alternative to using PBMC feeders. EXPERIMENTAL DESIGN We used aAPC or γ-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed based on pattern of T-cell receptor (TCR) usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. RESULTS The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, while increasing the frequency of CD8+ T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an up-regulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity and CD8+ T cells showed comparable anti-tumor function as those expanded with PBMC feeders. CONCLUSIONS TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical-application of aAPC to manufacture TIL for the treatment of melanoma. PMID:25304728

Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

PubMed

Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

2018-01-01

Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

Investigation of mitomycin-C-treated fibroblasts in 3-D collagen gel and conditioned medium for keratinocyte proliferation.

PubMed

Huang, Yi-Chau; Wang, Tzu-Wei; Sun, Jui-Sheng; Lin, Feng-Huei

2006-03-01

Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. In this study, the paracrine effect of mitomycin-C-treated fibroblasts with various densities in collagen gel for keratinocyte proliferation was investigated from which an optimum cell density and optimum conditioned medium would be determined to expand keratinocyte without further differentiation for skin equivalent tissue engineering. The optimum cell density in collagen feeder gel for optimum collected medium preparation will be determined by checking the level of keratinocyte growth factor and granulocyte macrophage colony-stimulating factor in conventional medium. The results showed that the cell density of 1 x 10(5) cells/gel in the feeder gel is better to produce optimum collected medium. The conditioned medium is prepared by mixing together the optimum collected medium and molecular cellular and developmental biology (MCDB) 153 medium in different ratios for keratinocyte growth. The keratinocyte viability will be measured by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed as the better medium for keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to coculture with keratinocytes is also determined as 1 x 10(5) cells/gel. Keratin 10 (K10) and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocytes should not be cultured in postconfluent conditions due to undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays a more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned medium could effectively promote proliferation, inhibit apoptosis, and prevent differentiation. The combination of conditioned media and feeder gel to culture keratinocytes without external supplements can provide an inexpensive way for keratinocyte proliferation and construct an environment for real-time communication between the two cells. The results conclude that keratinocyte cultivation in feeder gel with modified medium should be feasible in the production of high quality keratinocytes for skin equivalents preparation.

Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

Hydrodynamic control of phytoplankton loss to the benthos in an estuarine environment

USGS Publications Warehouse

Jones, Nicole L.; Thompson, Janet K.; Arrigo, Kevin R.; Monismith, Stephen G.

2009-01-01

Field experiments were undertaken to measure the influence of hydrodynamics on the removal of phytoplankton by benthic grazers in Suisun Slough, North San Francisco Bay. Chlorophyll a concentration boundary layers were found over beds inhabited by the active suspension feeders Corbula amurensis and Corophium alienense and the passive suspension feeders Marenzellaria viridis and Laonome sp. Benthic losses of phytoplankton were estimated via both the control volume and the vertical flux approach, in which chlorophyll a concentration was used as a proxy for phytoplankton biomass. The rate of phytoplankton loss to the bed was positively correlated to the bed shear stress. The maximum rate of phytoplankton loss to the bed was five times larger than estimated by laboratory-derived pumping rates for the active suspension feeders. Reasons for this discrepancy are explored including a physical mechanism whereby phytoplankton is entrained in a near-bed fluff layer where aggregation is mediated by the presence of mucus produced by the infaunal community.

Hydrodynamic control of phytoplankton loss to the benthos in an estuarine environment

USGS Publications Warehouse

Jones, N.L.; Thompson, J.K.; Arrigo, K.R.; Monismith, Stephen G.

2009-01-01

Field experiments were undertaken to measure the influence of hydrodynamics on the removal of phytoplankton by benthic grazers in Suisun Slough, North San Francisco Bay. Chlorophyll a concentration boundary layers were found over beds inhabited by the active suspension feeders Corbula amurensis and Corophium alienense and the passive suspension feeders Marenzellaria viridis and Laonome sp. Benthic losses of phytoplankton were estimated via both the control volume and the vertical flux approach, in which chlorophyll a concentration was used as a proxy for phytoplankton biomass. The rate of phytoplankton loss to the bed was positively correlated to the bed shear stress. The maximum rate of phytoplankton loss to the bed was five times larger than estimated by laboratory-derived pumping rates for the active suspension feeders. Reasons for this discrepancy are explored including a physical mechanism whereby phytoplankton is entrained in a near-bed fluff layer where aggregation is mediated by the presence of mucus produced by the infaunal community. ?? 2009, by the American Society of Limnology and Oceanography, Inc.

Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

PubMed Central

Lamb, Rebecca; Ambler, Carrie A.

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification. PMID:23326335

Evaluation of the Fretting Resistance of the High Voltage Insulation on the ITER Magnet Feeder Busbars

NASA Astrophysics Data System (ADS)

Clayton, N.; Crouchen, M.; Evans, D.; Gung, C.-Y.; Su, M.; Devred, A.; Piccin, R.

2017-12-01

The high voltage (HV) insulation on the ITER magnet feeder superconducting busbars and current leads will be prepared from S-glass fabric, pre-impregnated with an epoxy resin, which is interleaved with polyimide film and wrapped onto the components and cured during feeder manufacture. The insulation architecture consists of nine half-lapped layers of glass/Kapton, which is then enveloped in a ground-screen, and two further half-lapped layers of glass pre-preg for mechanical protection. The integrity of the HV insulation is critical in order to inhibit electrical arcs within the feeders. The insulation over the entire length of the HV components (bus bar, current leads and joints) must provide a level of voltage isolation of 30 kV. In operation, the insulation on ITER busbars will be subjected to high mechanical loads, arising from Lorentz forces, and in addition will be subjected to fretting erosion against stainless steel clamps, as the pulsed nature of some magnets results in longitudinal movement of the busbar. This work was aimed at assessing the wear on, and the changes in, the electrical properties of the insulation when subjected to typical ITER operating conditions. High voltage tests demonstrated that the electrical isolation of the insulation was intact after the fretting test.

GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

PubMed Central

Krtolica, Ana; Larocque, Nick; Genbacev, Olga; Ilic, Dusko; Coppe, Jean-Philippe; Patil, Christopher K.; Zdravkovic, Tamara; McMaster, Michael; Campisi, Judith; Fisher, Susan J.

2012-01-01

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ~6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical–basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical–basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation—βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell–cell contacts can trigger adoption of a neuronal fate. PMID:21396766

Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry.

PubMed

Peng, C A; Palsson, B Ø

1996-06-05

Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10(-1), 10(0), and 10(1)) and P(10(-2), 10(-1), 10(0), 10(1), and 10(2)). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, ..., 0.1 and Gz = 0.1, ..., 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions.

Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

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

Yue, Xiao-shan; Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501; Fujishiro, Masako

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells weremore » tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.« less

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

PubMed

Meng, G L; Zur Nieden, N I; Liu, S Y; Cormier, J T; Kallos, M S; Rancourt, D E

2008-04-01

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders. Copyright 2007 Wiley-Liss, Inc.

Human dermal papilla cells and outer root sheath cells: no follicular differentiation in nude mice and chicken embryos.

PubMed

Chiu, H C; Chang, C H; Jee, S H; Chang, C C; Wu, Y C

1994-09-01

Human scalp specimens were incubated in 5 U/ml dispase solution at 4 degrees C overnight before the isolation of dermal papillae and follicle epithelium. This pretreatment not only facilitated the attachment and cell outgrowth of dermal papillae but also made it easier to pluck out hairs with intact follicle epithelium. The outer root sheath cells were released from the follicle epithelium and grown on a feeder layer of mitomycin C-treated human dermal fibroblasts. The subcultured outer root sheath cells were grown in a serum-free medium. When the mixtures of early-passage dermal papilla cells and outer root sheath cells were injected into the subcutis of nude mice, an epidermal cyst surrounded by layers of fibrous tissue was found in three weeks. No hair follicles were found when the mixtures were implanted onto the chorioallantoic membrane of nine-day-old chicken embryos. A keratinized mass lying on the chorionic epithelium with or without smaller similar masses in the chorioallantoic membrane was found in eight days. No hair follicle-like structure could be found. Possible factors contributing to the failure to undergo follicular differentiation in this study are discussed.

The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with Mesenchymal Stem Cells.

PubMed

Oubari, Farhad; Amirizade, Naser; Mohammadpour, Hemn; Nakhlestani, Mozhdeh; Zarif, Mahin Nikougoftar

2015-01-01

Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+(CD34(+)) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05). FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture.

Mapping the 3-D extent of the Northern Lobe of the Bushveld layered mafic intrusion from geophysical data

USGS Publications Warehouse

Finn, Carol A.; Bedrosian, Paul A.; Cole, Janine; Khoza, Tshepo David; Webb, Susan J.

2015-01-01

Geophysical models image the 3D geometry of the mafic portion of the Bushveld Complex north of the Thabazimbi-Murchison Lineament (TML), critical for understanding the origin of the world's largest layered mafic intrusion and platinum group element deposits. The combination of the gravity and magnetic data with recent seismic, MT, borehole and rock property measurements powerfully constrains the models. The intrusion north of the TML is generally shallowly buried (generally <1500 m) with a modeled area of ∼160 km × ∼125 km. The modeled thicknesses are not well constrained but vary from ∼<1000 to >12,000 m, averaging ∼4000 m. A feeder, suggested by a large modeled thickness (>10,000 m) and funnel shape, for Lower Zone magmas could have originated near the intersection of NS and NE trending TML faults under Mokopane. The TML has been thought to be the feeder zone for the entire Bushveld Complex but the identification of local feeders and/or dikes in the TML in the models is complicated by uncertainties on the syn- and post-Bushveld deformation history. However, modeled moderately thick high density material near the intersection of faults within the central and western TML may represent feeders for parts of the Bushveld Complex if deformation was minimal. The correspondence of flat, high resistivity and density regions reflect the sill-like geometry of the Bushveld Complex without evidence for feeders north of Mokopane. Magnetotelluric models indicate that the Transvaal sedimentary basin underlies much of the Bushveld Complex north of the TML, further than previously thought and important because the degree of reaction and assimilation of the Transvaal rocks with the mafic magmas resulted in a variety of mineralization zones.

Comparison of the canine corneal epithelial cell sheets cultivated from limbal stem cells on canine amniotic membrane, atelocollagen gel, and temperature-responsive culture dish.

PubMed

Nam, Eunryel; Fujita, Naoki; Morita, Maresuke; Tsuzuki, Keiko; Lin, Hsing Yi; Chung, Cheng Shu; Nakagawa, Takayuki; Nishimura, Ryohei

2015-07-01

The current study compared canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane, atelocollagen gel, and temperature-responsive culture dish. We collected limbal epithelial cells from the intact eyes of beagles and cultivated the cells on denuded canine amniotic membranes, temperature-responsive cell culture labware, and collagen gel with 3T3 feeder cells. Immunofluorescence staining for Ki-67 was used to analyze the capacity of cell proliferation in the sheets. Immunofluorescence staining was also performed for the corneal epithelium-specific marker cytokeratin 3 and putative stem cell markers ABCG2 and p63. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect ABCG2 and p63. The growth rates of the cultivated cells, or the times it took them to reach confluency, were different for the three scaffolds. The cultivated sheet on the temperature-responsive dish consisted of 2-3 layers, while those on the collagen gel and on the amniotic membrane consisted of 5-8 layers. The basal layer cells grown on all three scaffolds expressed putative stem cell markers. In real-time RT-PCR analysis, the highest level of p63 was observed in the sheets grown on collagen gel. In this study, the cells cultured on the collagen gel demonstrated a capacity for cell proliferation, and the expressions of stem cells in the sheets suggested that collagen gel is the most suitable carrier for clinical use. © 2014 American College of Veterinary Ophthalmologists.

Rapid fibroblast removal from high density human embryonic stem cell cultures.

PubMed

Turner, William S; McCloskey, Kara E

2012-10-28

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation(1). This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity(4). Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types(5-8)). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Release of infectious cells from epidermal ulcers in Ichthyophonus sp.–infected Pacific Herring (Clupea pallasii): Evidence for multiple mechanisms of transmission

USGS Publications Warehouse

Hershberger, Paul K.; Gregg, Jacob L.; Kocan, R.M.

2010-01-01

A common clinical sign of ichthyophoniasis in herring and trout is “sandpaper” skin, a roughening of the epidermis characterized by the appearance of small papules, followed by ulceration and sloughing of the epithelium; early investigators hypothesized that these ulcers might be a means of transmitting the parasite, Ichthyophonus sp., without the necessity of ingesting an infected host. We examined the cells associated with the epidermal lesions and confirmed that they were viable Ichthyophonus sp. cells that were readily released from the skin into the mucous layer and ultimately into the aquatic environment. The released cells were infectious when injected into the body cavity of specific-pathogen-free herring. Our hypothesis is that different mechanisms of transmission occur in carnivorous and planktivorous hosts: Planktonic feeders become infected by ingestion of ulcer-derived cells, while carnivores become infected by ingestion of whole infected fish.

A new system for cultivation of human keratinocytes on acellular dermal matrix substitute with the use of human fibroblast feeder layer.

PubMed

Xiao, S; Zhu, S; Ma, B; Xia, Z-F; Yang, J; Wang, G

2008-01-01

To improve the proliferative potential of human keratinocytes (HK) cultured on acellular dermal matrix (ADM), HK and mitomycin C-treated human fibroblasts (MMC-HF) were seeded onto ADM to form four types of composite skin: type I, HK were seeded onto the epidermal side of ADM; type II, both HK and MMC-HF were seeded onto the epidermal side; type III, MMC-HF were preseeded onto the dermal side of ADM, and then HK were seeded onto the epidermal side, and type IV, where MMC-HF were preseeded onto both sides, and then HK were seeded onto the epidermal side. Compared with type I and III, the proliferative potential of HK of type II and IV was significantly higher on day 3, 5, 7 and 9 in vitro. In type I and III, HK grew into one layer on day 7-9, while in type II and IV keratinocytes grew into a confluent monolayer by day 4-6. The adherence to ADM of HK in types II and IV was stronger than that in type I and III. The take rate of type II and IV composite skin was also significantly higher. In conclusion, when MMC-HF and HK were cocultured on the epidermal side of ADM, MMC-HF could serve as excellent feeder cells. Copyright 2007 S. Karger AG, Basel.

Mechanism of induction of fibroblast to corneal endothelial cell.

PubMed

Jiang, Yan; Fu, Wei-Cai; Zhang, Lin

2014-08-01

To explore mechanism of nduction of fibroblast to corneal endothelial cell. Rabbit conjunctiva fibroblasts were used as feeder cells, rabbit oral mucosa epithelial cells were used as seed cells, and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium. The transformation effect was observed. As concentration of mitomycin C increased, cell survival rate gradually decreased, cell proliferation was obviously inhibited when concentration≥25 μg/mL; 5 days after being treated by 5 μg/mL mitomycin C, cell body was enlarged and extended without cell fusion, however after being treated by 0.5 μg/mL mitomycin C, cell body was significantly proliferated and gradually fused; after 3 weeks of culture, stratified epithelium appeared on rabbit oral mucosa epithelial cells, differentiation layers were 4-5 and were well differentiated, the morphology was similar to corneal endothelial cells; Under electron microscope, surface layer of cells were polygonal, tightly connected to another with microvilli on the border, there was hemidesmosome between basal cells and human denuded amniotic membrane. Fibroblast cells have the potential of multi-directional differentiation, effective induction can promote emergence of intercellular desmosomes between seed cells and emergence of epithelial surface microvilli, and differentiate to the corneal endothelial cell. However, clinical application still needs more research and safety evaluation. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

PubMed Central

Yanagimachi, Masakatsu D.; Niwa, Akira; Tanaka, Takayuki; Honda-Ozaki, Fumiko; Nishimoto, Seiko; Murata, Yuuki; Yasumi, Takahiro; Ito, Jun; Tomida, Shota; Oshima, Koichi; Asaka, Isao; Goto, Hiroaki; Heike, Toshio; Nakahata, Tatsutoshi; Saito, Megumu K.

2013-01-01

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×106±0.3×106 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery. PMID:23573196

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

PubMed

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Establishment and characterization of feeder cell-dependent bovine fetal liver cell lines.

PubMed

Talbot, Neil C; Wang, Ling; Garrett, Wesley M; Caperna, Thomas J; Tang, Young

2016-03-01

The establishment and initial characterization of bovine fetal liver cell lines are described. Bovine fetal hepatocytes were cultured from the liver of a 34-d bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO (SIMS mouse strain, thioguanine- and ouabain-resistant) feeder layers and were cultured in a medium supplemented with 10% fetal bovine serum. After 2-3 wk, primary colonies of hepatocytes were observed by phase-contrast microscopic observation. Individual hepatocyte colonies were colony-cloned into independent bovine fetal liver (BFL) cell lines. Two cell lines, BFL-6 and BFL-9, grew the best of several isolates, and they were further characterized for growth potential and for hepatocyte morphology and function. The two cell lines were found to grow markedly better in the presence of the transforming growth factor (TGF)-beta inhibitor, SB431542 (1 μM). Their continuous culture also depended on a particular medium height-for T12.5 flasks, 3 ml total medium produced optimum growth. Higher or lower amounts of medium caused less cell growth or cessation of growth. The cell lines were propagated for over a year at split ratios of 1:2 or 1:3 at each passage until reaching senescence at approximately 30 passages. The cells were laterally polarized with well-developed canalicular spaces occurring between adjacent BFL cells. Treatment of the cultures with cyclic adenosine monophosphate (cAMP)-stimulating chemicals or peptides (e.g., forskolin or glucagon) caused physical expansion of the canaliculi between the cells within 15 min. The cells secreted a spectrum of serum proteins, were positive for the expression of several hepatocyte-specific genes, and converted ammonia to urea, although at a relatively low rate. The culture system provides an in vitro model of fetal bovine hepatocytes and is the first demonstration of the continuous culture of normal bovine hepatocytes as cell lines.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

PubMed

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

PubMed

Panda, Santanu; Bisht, Sonu; Malakar, Dhruba; Mohanty, Ashok K; Kaushik, Jai K

2015-01-01

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes. Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66)×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies. We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.

Difference in suitable mechanical properties of three-dimensional, synthetic scaffolds for self-renewing mouse embryonic stem cells of different genetic backgrounds.

PubMed

Lee, Myungook; Ahn, Jong Il; Ahn, Ji Yeon; Yang, Woo Sub; Hubbell, Jeffrey A; Lim, Jeong Mook; Lee, Seung Tae

2017-11-01

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017. © 2016 Wiley Periodicals, Inc.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

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

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Organogenesis from transformed tomato explants.

PubMed

Frary, Anne; Van Eck, Joyce

2005-01-01

Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants. Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development. In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer. The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation. After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin. Regenerated shoots are then rooted on a separate selective medium. This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10-15%.

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity.

PubMed

Higuchi, Akon; Kao, Shih-Hsuan; Ling, Qing-Dong; Chen, Yen-Ming; Li, Hsing-Fen; Alarfaj, Abdullah A; Munusamy, Murugan A; Murugan, Kadarkarai; Chang, Shih-Chang; Lee, Hsin-Chung; Hsu, Shih-Tien; Kumar, S Suresh; Umezawa, Akihiro

2015-12-14

The tentative clinical application of human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human induced pluripotent stem cells, is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore, we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

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

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

PubMed

Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

2015-04-01

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro.

PubMed

Tang, Patrick Ming-Kuen; Tang, Philip Chiu-Tsun; Chung, Jeff Yat-Fai; Hung, Jessica Shuk Chun; Wang, Qing-Ming; Lian, Guang-Yu; Sheng, Jingyi; Huang, Xiao-Ru; To, Ka-Fai; Lan, Hui-Yao

2018-01-09

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

Maintaining the pluripotency of mouse embryonic stem cells on gold nanoparticle layers with nanoscale but not microscale surface roughness

NASA Astrophysics Data System (ADS)

Lyu, Zhonglin; Wang, Hongwei; Wang, Yanyun; Ding, Kaiguo; Liu, Huan; Yuan, Lin; Shi, Xiujuan; Wang, Mengmeng; Wang, Yanwei; Chen, Hong

2014-05-01

Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01540a

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

Maintaining the pluripotency of mouse embryonic stem cells on gold nanoparticle layers with nanoscale but not microscale surface roughness.

PubMed

Lyu, Zhonglin; Wang, Hongwei; Wang, Yanyun; Ding, Kaiguo; Liu, Huan; Yuan, Lin; Shi, Xiujuan; Wang, Mengmeng; Wang, Yanwei; Chen, Hong

2014-06-21

Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.

Theileria parva infection induces autocrine growth of bovine lymphocytes.

PubMed Central

Dobbelaere, D A; Coquerelle, T M; Roditi, I J; Eichhorn, M; Williams, R O

1988-01-01

Bovine lymphocytes infected with the parasite Theileria parva continuously secrete a growth factor that is essential for their proliferation in vitro and also constitutively express interleukin 2 receptors on their surface. Dilution of the secreted growth factor, caused by culturing cells at low density, results in retardation of culture growth. Human recombinant interleukin 2, however, effectively substitutes for the diluted growth factor by restoring normal growth rates and also allows Theileria-infected cells to be grown at low density without the use of feeder layers. Secretion of the growth factor and expression of the interleukin 2 receptor depend on the presence of the parasite in the cytoplasm of the host cell. Elimination of the parasite from the cell cytoplasm by the specific antitheilerial drug BW 720c results in the arrest of growth factor secretion and the disappearance of interleukin 2 receptors from the cell surface. This is accompanied by growth arrest and reversion of the infected cells to the morphology of resting lymphocytes. We propose that the continuous proliferation of infected cells in vitro is mediated by autocrine receptor activation. Images PMID:3133661

In vitro study of cell differentiation by two type mouse embryo stem cells on mono- and multilayer nanocarbon tubes

NASA Astrophysics Data System (ADS)

Imai, Koichi; Akasaka, Tsukasa; Watari, Fumio; Tanoue, Akito; Nakamura, Kazuaki; Suese, Kazuhiko; Takashima, Hiromasa; Nishikawa, Tetsunari; Tanaka, Akio; Takeda, Shoji

2012-09-01

The effects of nanomaterials on human reproduction and development remain unknown. The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We previously investigated the myocardial cell differentiation of ES-D3 cells using monolayer (SWCNTs) and multilayer (MWCNTs) nanocarbon tubes. As a result, in spite of having the same carbon composition, the effects on the cell differentiation levels differed between the tubes. We investigated their cell differentiation and cytotoxic effects on EL M3 and ES-R1-EGFP B2/EGFP cells, which require feeder cells. As a result, myocardial pulse rates differed between the presence of SWCNTs and MWCNTs even when feeder cells existed between the samples and cells. The different surface structures of SWCNTs and MWCNTs may have influenced ES cell differentiation.

CFD Application to Flow-Accelerated Corrosion in Feeder Bends

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

Pietralik, John M.; Smith, Bruce A.W.

2006-07-01

Feeder piping in CANDU{sup R} plants experiences a thinning degradation mechanism called Flow-Accelerated Corrosion (FAC). The piping is made of carbon steel and has high water flow speeds. Although the water chemistry is highly alkaline with room-temperature pH in a range of 10.0-10.5, the piping has FAC rates exceeding 0.1 mm/year in some locations, e.g., in bends. One of the most important parameters affecting the FAC rate is the mass transfer coefficient for convective mass transport of ferrous ions. The ions are created at the pipe wall as a result of corrosion, diffuse through the oxide layer, and are transportedmore » from the oxide-layer/water interface to the bulk water by mass transport. Consequently, the local flow characteristics contribute to the highly turbulent convective mass transfer. Plant data and laboratory experiments indicate that the mass transfer step dominates FAC under feeder conditions. In this study, the flow and mass transfer in a feeder bend under operating conditions were simulated using the Fluent{sup TM} computer code. Because the flow speed is very high, with the Reynolds numbers in a range of several millions, and because the geometry is complex, experiments in a 1:1 scale were conducted with the main objective to validate flow simulations. The experiments measured pressure at several key locations and visualized the flow. The flow and mass transfer models were validated using available friction-factor and mass transfer correlations and literature experiments on mass transfer in a bend. The validation showed that the turbulence model that best predicts the experiments is the realizable k-{epsilon} model. Other two-equation turbulence models, as well as one-equation models and Reynolds stress models were tried. The near-wall treatment used the non-equilibrium wall functions. The wall functions were modified for surface roughness when necessary. A comparison of the local mass transfer coefficient with measured FAC rate in plant specimens shows very good agreement. Visualization experiments indicate secondary flows in the bends. No boundary layer separation was observed in experiments or in simulations. (authors)« less

A single-cell and feeder-free culture system for monkey embryonic stem cells.

PubMed

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

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

Aslanova, Afag; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666; Takagi, Ryo

With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferationmore » of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen transmission that might arise from animal-derived materials. - Highlights: • Y-27632 promotes the proliferation of human keratinocytes. • Human keratinocytes with Y-27632 can stratify similarly to traditional method. • Y-27632 is useful for culture medium of human keratinocyte in clinical setting.« less

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

PubMed Central

Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N.; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao

2015-01-01

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10–50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

Utility of human hepatocyte spheroids without feeder cells for evaluation of hepatotoxicity.

PubMed

Ogihara, Takuo; Arakawa, Hiroshi; Jomura, Tomoko; Idota, Yoko; Koyama, Satoshi; Yano, Kentaro; Kojima, Hajime

2017-01-01

We investigated the utility of three-dimensionally cultured hepatocytes (spheroids) without feeder cells (Sph(f-)) for the prediction of drug-induced liver injury (DILI) in humans. Sph(f-) and spheroids cultured on feeder cells (Sph(f+)) were exposed to the hepatotoxic drugs flutamide, diclofenac, isoniazid and chlorpromazine at various concentrations for 14 days, and albumin secretion and cumulative leakages of toxicity marker enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (γ-GTP), were measured. The cumulative AST, LDH or γ-GTP leakages from Sph(f-) were similar to or greater than those from Sph(f+) for all drugs tested, although ALT leakages showed no consistent difference between Sph(f+) and Sph(f-). In the case of Sph(f-), significant correlations among all the toxicity markers except for γ-GTP were observed. As regards the drug concentrations causing 1.2-fold elevation of enzyme leakage (F 1.2 ), no consistent difference between Sph(f+) and Sph(f-) was found, although several F 1.2 values were undetermined, especially in Sph(f+). The IC 50 of albumin secretion and F 1.2 of AST leakage from Sph(f-) were equal to or lower than those of Sph(f+) for all the tested drugs. These results indicate that feeder cells might contribute to resistance to hepatotoxicity, suggesting DILI could be evaluated more accurately by using Sph(f-). We suggest that long-term exposure of Sph(f-) to drugs might be a versatile method to predict and reproduce clinical chronic toxicity, especially in response to repeated drug administration.

Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression.

PubMed

Yuri, Shunsuke; Fujimura, Sayoko; Nimura, Keisuke; Takeda, Naoki; Toyooka, Yayoi; Fujimura, Yu-Ichi; Aburatani, Hiroyuki; Ura, Kiyoe; Koseki, Haruhiko; Niwa, Hitoshi; Nishinakamura, Ryuichi

2009-04-01

Sall4 is a mouse homolog of a causative gene of the autosomal dominant disorder Okihiro syndrome. We previously showed that the absence of Sall4 leads to lethality during peri-implantation and that Sall4-null embryonic stem (ES) cells proliferate poorly with intact pluripotency when cultured on feeder cells. Here, we report that, in the absence of feeder cells, Sall4-null ES cells express the trophectoderm marker Cdx2, but are maintained for a long period in an undifferentiated state with minimally affected Oct3/4 expression. Feeder-free Sall4-null ES cells contribute solely to the inner cell mass and epiblast in vivo, indicating that these cells still retain pluripotency and do not fully commit to the trophectoderm. These phenotypes could arise from derepression of the Cdx2 promoter, which is normally suppressed by Sall4 and the Mi2/NuRD HDAC complex. However, proliferation was impaired and G1 phase prolonged in the absence of Sall4, suggesting another role for Sall4 in cell cycle control. Although Sall1, also a Sall family gene, is known to genetically interact with Sall4 in vivo, Sall1-null ES cells have no apparent defects and no exacerbation is observed in ES cells lacking both Sall1 and Sall4, compared with Sall4-null cells. This suggests a unique role for Sall4 in ES cells. Thus, though Sall4 does not contribute to the central machinery of the pluripotency, it stabilizes ES cells by repressing aberrant trophectoderm gene expression.

Generation of Human Induced Pluripotent Stem Cells Using RNA-Based Sendai Virus System and Pluripotency Validation of the Resulting Cell Population.

PubMed

Chichagova, Valeria; Sanchez-Vera, Irene; Armstrong, Lyle; Steel, David; Lako, Majlinda

2016-01-01

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.

In Vitro Evaluation of Cell-Seeded Chitosan Films for Peripheral Nerve Tissue Engineering

PubMed Central

Wrobel, Sandra; Serra, Sofia Cristina; Ribeiro-Samy, Silvina; Sousa, Nuno; Heimann, Claudia; Barwig, Christina; Grothe, Claudia; Haastert-Talini, Kirsten

2014-01-01

Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)—immortalized, neonatal, and adult—as well as rat bone-marrow-derived mesenchymal stromal cells (BMSCs) were analyzed with regard to their cell metabolic activity, proliferation profiles, and cell morphology after different time points of mono- and cocultures on the chitosan films. Overall the results demonstrate a good cytocompatibility of the chitosan substrate. Both cell types were viable on the biomaterial and showed different metabolic activities and proliferation behavior, indicating cell-type-specific cell–biomaterial interaction. Moreover, the cell types also displayed their typical morphology. In cocultures adult SCs used the BMSCs as a feeder layer and no negative interactions between both cell types were detected. Further, the chitosan films allow neurite outgrowth from dissociated sensory neurons, which is additionally supported on film preseeded with SC-BMSC cocultures. The presented chitosan films therefore demonstrate high potential for their use in tissue-engineered nerve grafts. PMID:24606318

Layered double hydroxide nanoparticles promote self-renewal of mouse embryonic stem cells through the PI3K signaling pathway

NASA Astrophysics Data System (ADS)

Wu, Youjun; Zhu, Rongrong; Zhou, Yang; Zhang, Jun; Wang, Wenrui; Sun, Xiaoyu; Wu, Xianzheng; Cheng, Liming; Zhang, Jing; Wang, Shilong

2015-06-01

Embryonic stem cells (ESCs) hold great potential for regenerative medicine due to their two unique characteristics: self-renewal and pluripotency. Several groups of nanoparticles have shown promising applications in directing the stem cell fate. Herein, we investigated the cellular effects of layered double hydroxide nanoparticles (LDH NPs) on mouse ESCs (mESCs) and the associated molecular mechanisms. Mg-Al-LDH NPs with an average diameter of ~100 nm were prepared by hydrothermal methods. To determine the influences of LDH NPs on mESCs, cellular cytotoxicity, self-renewal, differentiation potential, and the possible signaling pathways were explored. Evaluation of cell viability, lactate dehydrogenase release, ROS generation and apoptosis demonstrated the low cytotoxicity of LDH NPs. The alkaline phosphatase activity and the expression of pluripotency genes in mESCs were examined, which indicated that exposure to LDH NPs could support self-renewal and inhibit spontaneous differentiation of mESCs under feeder-free culture conditions. The self-renewal promotion was further proved to be independent of the leukemia inhibitory factor (LIF). Furthermore, cells treated with LDH NPs maintained the potential to differentiate into all three germ layers both in vitro and in vivo through formation of embryoid bodies and teratomas. In addition, we observed that LDH NPs initiated the activation of the PI3K/Akt pathway, while treatment with the PI3K inhibitor LY294002 could block the effects of LDH NPs on mESCs. The results confirmed that the promotion of self-renewal by LDH NPs was associated with activation of the PI3K/Akt signaling pathway. Altogether, our studies identified a new role of LDH NPs in maintaining self-renewal of mouse ES cells which could potentially be applied in stem cell research.Embryonic stem cells (ESCs) hold great potential for regenerative medicine due to their two unique characteristics: self-renewal and pluripotency. Several groups of nanoparticles have shown promising applications in directing the stem cell fate. Herein, we investigated the cellular effects of layered double hydroxide nanoparticles (LDH NPs) on mouse ESCs (mESCs) and the associated molecular mechanisms. Mg-Al-LDH NPs with an average diameter of ~100 nm were prepared by hydrothermal methods. To determine the influences of LDH NPs on mESCs, cellular cytotoxicity, self-renewal, differentiation potential, and the possible signaling pathways were explored. Evaluation of cell viability, lactate dehydrogenase release, ROS generation and apoptosis demonstrated the low cytotoxicity of LDH NPs. The alkaline phosphatase activity and the expression of pluripotency genes in mESCs were examined, which indicated that exposure to LDH NPs could support self-renewal and inhibit spontaneous differentiation of mESCs under feeder-free culture conditions. The self-renewal promotion was further proved to be independent of the leukemia inhibitory factor (LIF). Furthermore, cells treated with LDH NPs maintained the potential to differentiate into all three germ layers both in vitro and in vivo through formation of embryoid bodies and teratomas. In addition, we observed that LDH NPs initiated the activation of the PI3K/Akt pathway, while treatment with the PI3K inhibitor LY294002 could block the effects of LDH NPs on mESCs. The results confirmed that the promotion of self-renewal by LDH NPs was associated with activation of the PI3K/Akt signaling pathway. Altogether, our studies identified a new role of LDH NPs in maintaining self-renewal of mouse ES cells which could potentially be applied in stem cell research. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02339d

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

PubMed

Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

2015-11-01

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

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

Yu, Lingling; Noncoding RNA Center, Yangzhou University, Yangzhou 225001; Zhao, Yingmin

Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feedermore » layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.« less

Granular Material Scoop and Near-Vertical Lifting Feeder/Conveyor

NASA Technical Reports Server (NTRS)

Walton, Otis (Inventor); Vollmer, Hubert J. (Inventor)

2017-01-01

An integrated granular-material scoop and near-vertical lifting feeder/conveyor includes special connections and skirts between a bullnose rotating scoop and an open-helical screw that provides the rotations and material lift and evacuation. A conical working-face of the bullnose rotating scoop has symmetrically distributed graters and vents to break loose and force-in granular material from natural deposits and cargo holds. The bullnose rotating scoop and the open-helical screw its attached to move the material into a continuous layer on the inside surface of an outer stationary sheathing. A motor drive attached to the open-helical screw above at the delivery end provides the lifting force necessary.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

PubMed

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

PubMed Central

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system. PMID:24465853

Ultrastructure of the post-corpus of Zeldia punctata (Cephalobina) for analysis of the evolutionary framework of nematodes related to Caenorhabditis elegans (Rhabditina).

PubMed Central

Zhang, Y C; Baldwin, J G

2000-01-01

The ultrastructure of the post-corpus of Zeldia punctata (Cephalobina) was compared with previous observations of Caenorhabditis elegans (Rhabditina) and Diplenteron sp. (Diplogastrina) with the goal of interpreting the morphological evolution of the feeding structures in the Secernentea. The post-corpus of Z. punctata consists of six marginal, 13 muscle, five gland and seven nerve cells. The most anterior of four layers of muscle cells consists of six mononucleate cells in Z. punctata. The homologous layer in C. elegans and Diplenteron consists of three binucleate cells, suggesting a unique derived character (synapomorphy) shared between the Rhabditina and Diplogastrina. Contrary to Diplenteron sp. where we observed three oesophageal glands, Z. punctata and C. elegans have five oesophageal glands. We question this shared character as reflecting a common evolution between the Cephalobina and Rhabditina, because there are strong arguments for functional (adaptive) convergence of the five glands in these bacterial feeders. Convergence is further suggested by the mosaic distribution of three versus five glands throughout the Nemata; this distribution creates difficulties in establishing character polarity. Although morphological data are often laborious to recover and interpret, we nevertheless view 'reciprocal illumination' between molecular and morphological characters as the most promising and robust process for reconstructing the evolution of the Secernentea and its feeding structures. PMID:10902689

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa.

PubMed

Zofou, Denis; Fombad, Fanny Fri; Gandjui, Narcisse V T; Njouendou, Abdel Jelil; Kengne-Ouafo, Arnaud Jonas; Chounna Ndongmo, Patrick W; Datchoua-Poutcheu, Fabrice R; Enyong, Peter A; Bita, Dizzle Tayong; Taylor, Mark J; Turner, Joseph D; Wanji, Samuel

2018-05-02

Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T 90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T 90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T 90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility. The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

PubMed Central

Laugwitz, Karl-Ludwig; Moretti, Alessandra; Lam, Jason; Gruber, Peter; Chen, Yinhong; Woodard, Sarah; Lin, Li-Zhu; Cai, Chen-Leng; Lu, Min Min; Reth, Michael; Platoshyn, Oleksandr; Yuan, Jason X.-J.; Evans, Sylvia; Chien, Kenneth R.

2017-01-01

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases1–3. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart4. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease. PMID:15703750

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

PubMed

Lidgerwood, Grace E; Lim, Shiang Y; Crombie, Duncan E; Ali, Ray; Gill, Katherine P; Hernández, Damián; Kie, Josh; Conquest, Alison; Waugh, Hayley S; Wong, Raymond C B; Liang, Helena H; Hewitt, Alex W; Davidson, Kathryn C; Pébay, Alice

2016-04-01

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.

Identification of pectenotoxins in plankton, filter feeders, and isolated cells of a Dinophysis acuminata with an atypical toxin profile, from Chile.

PubMed

Blanco, Juan; Alvarez, Gonzalo; Uribe, Eduardo

2007-04-01

A bloom of Dinophysis acuminata produced, in autumn of 2005, a closure of the scallop harvesting in Bahía Inglesa, in the Chilean III region. Isolated cells of this Dinophysis species were shown to contain 180 pg cell(-1) of pectenotoxin 2 but neither okadaic acid nor any of its analogs or derivatives (at least at a detectable level). Examination of plankton and filter-feeder samples covering an area of ca. 350 km, from the location where the toxicity was recorded to Bahía Tongoy, showed that the unique toxin profile found in the first bloom was widespread over that part of Chile and persisted for months. The analysis were carried out by HPLC-ESI-MS using positive ionization mode, with a detection limit below 2 ng of OA mL(-1) of methanolic extract. This is the first report of the presence of pectenotoxins in the plankton of the Pacific coast of America and in the studied filter feeders. This is also the first report of a Dinophysis species containing pectenotoxins and not any toxin of the okadaic acid group.

Design and characterization of hybrid peptide sol-gel materials for the solid state induction of neuronal differentiation

NASA Astrophysics Data System (ADS)

Jedlicka, Sabrina S.

2007-12-01

Cell-based therapeutics are a rapidly growing area of research, with considerable promise in the treatment of neurological diseases. One of the primary limitations to neuronal cell-based devices is the necessity to maintain cells in an immature or undifferentiated state in culture prior to transplantation. In many cases, the undifferentiated cell does not express the desired characteristics for implantation. Biologically functional nanomaterials provide the ability to manipulate the direct extracellular environment surrounding cells; influencing their fate and differentiation path. The ability to engineer the interface between the cells and culture materials provides a repeatable, stable means of directing cells down a specific growth path determined by endogenous signaling pathways. This materials approach to cellular engineering can limit the need for added exogenous growth factors, "feeder" layers, or animal sera, in addition to creating a homogenous cell population for transplantation. In this work, hybrid peptide ormosil materials were developed; designed to mimic the developing mammalian brain during corticogenesis. These materials have been developed to enhance the GABAergic phenotype of P19 embryonic carcinoma cells and immature immortalized neurons. The ability to develop a homogenous, directed cell population has implications in stem cell research, regenerative medicine, cell-based devices and biosensing technology.

Determination of body width in brown and white layer pullets by image analyses.

PubMed

Giersberg, M F; Kemper, N; Hartung, J; Schrader, L; Spindler, B

2017-06-01

1. Specific legal requirements for keeping pullets are not available in the European Union. However, two of the most important rearing factors for pullets are sufficient perching and feeder space. Both factors represent horizontal space dimensions which derive from the body width of the birds. 2. The body width of two strains of layer pullets (brown (BL) and white (WL) layer pullets) based on the measurement of distances in digital images was conducted on front-view digital photographs of BL and WL pullets taken at 8, 12 and 19 weeks of life. 3. Depending on live weight, age and body position, BL pullets measured an average body width between 10.70 ± 1.10 and 13.96 ± 1.11 cm. The width of WL pullets ranged from 10.30 ± 0.86 to 13.00 ± 1.14 cm. 4. Compared with WL, BL pullets occupied more horizontal space during rearing. Age influenced the body width of BL and WL pullets at the end of rearing. The tested body positions of the pullets did not affect the measured body width. 5. The biometric data obtained in this study are a useful basis for developing legal requirements for pullets, especially for defining minimum perch width and feeder space allowances.

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells

PubMed Central

Nakagawa, Masato; Taniguchi, Yukimasa; Senda, Sho; Takizawa, Nanako; Ichisaka, Tomoko; Asano, Kanako; Morizane, Asuka; Doi, Daisuke; Takahashi, Jun; Nishizawa, Masatoshi; Yoshida, Yoshinori; Toyoda, Taro; Osafune, Kenji; Sekiguchi, Kiyotoshi; Yamanaka, Shinya

2014-01-01

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system. PMID:24399248

Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

PubMed

Trokovic, Ras; Weltner, Jere; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Salomaa, Veikko; Jalanko, Anu; Otonkoski, Timo; Kyttälä, Aija

2014-12-01

Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking. ©AlphaMed Press.

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

USDA-ARS?s Scientific Manuscript database

The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

PubMed

Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

2017-06-08

Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

Rational design of bottom blocks for development of ore deposits systems with caving of ore and enclosing rocks

NASA Astrophysics Data System (ADS)

Versilov, S. O.; Posylniy, Yu V.; Shurygin, D. N.; Tretyak, A. Ya

2017-10-01

The assessment of the geological conditions of development of existing ore deposits was made. For testing ore deposits in difficult mining and geological conditions, the authors proposed the system of development, accompanied by collapse of the mechanical ore with the use of feeders of active action that could be manufactured directly in the mine in accordance with the specific conditions of occurrence of minerals. The paper demonstrates the technology of manufacture of load-bearing structures of the feeder directly in the mine at the scene of the breaking of the first layer of ore, as well as the dynamics of the ore and the choice of parameters of concrete feeders. A new design of the bottom block was proposed, the idea of technical solution of which consists in the fact that it is offered to undergo the production of the smallest possible cross section, which is determined only by the dimensions of the conveyors to deliver ore. And before the explosion of fans of production wells, it is necessary to produce local collapse of the roof production to increase its height at the place of production of ore by blasting wellheads in two or three rows.

A Simple and Robust Method for Culturing Human-Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin.

PubMed

Kim, Mee-Hae; Matsubara, Yoshifumi; Fujinaga, Yukako; Kino-Oka, Masahiro

2018-02-01

Clinical and industrial applications of human-induced pluripotent stem cells (hiPSCs) is hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, a simple and robust hiPSC-culture-propagation strategy incorporating botulinum hemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state is developed. After HA treatment, cell-cell adhesion is disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. The authors find that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA are confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintains expression of pluripotent markers and readily forms embryoid bodies expressing markers for all three germ-cell layers. This method enables highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.

PubMed

Medrano, Jose V; Rombaut, Charlotte; Simon, Carlos; Pellicer, Antonio; Goossens, Ellen

2016-11-01

To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA - )/epithelial cell surface antigen (EPCAM + ) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA - /EPCAM + resulted in an enrichment of 27% VASA + /UTF1 + hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm 2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm 2 ) and differentially plated cells (49 hSSCS/cm 2 ). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA - /EPCAM + sorted cells with testicular feeders improved the germ cell/somatic cell ratio. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Trace-fossil assemblages with a new ichnogenus in "spotted"

NASA Astrophysics Data System (ADS)

Šimo, Vladimír; Tomašových, Adam

2013-10-01

Highly-bioturbated "spotted" limestones and marls (Fleckenmergel-Fleckenkalk facies) of the Early Jurassic, which were deposited in broad and recurrent deep-shelf habitats of the Northern Tethys, are characterized by rare benthic carbonate-producing macroinvertebrates. To address this paradox, we analyse trace-fossil assemblages in a ~85 m-thick succession of Pliensbachian spotted deposits (Zliechov Basin, Western Carpathians). They are dominated by infaunal and semi-infaunal deposit-feeders, with 9 ichnogenera and pyritized tubes of the semi-infaunal foraminifer Bathysiphon, being dominated by Chondrites, Lamellaeichnus (new ichnogenus), and Teichichnus. Lamellaeichnus, represented by a horizontal basal cylindrical burrow and an upper row of stacked convex-up gutters, was produced by a mobile deposit-feeder inhabiting shallow tiers because it is crossed by most other trace fossils. We show that the spotty appearance of the deposits is generated by a mixture of (1) dark, organic-rich shallow- and deep-tier traces (TOC = 0.16-0.36), and (2) light grey, organic-poor mottled or structurless sediment (TOC = 0.09-0.22). The higher TOC in shallow-tier burrows of Lamellaeichnus demonstrates that uppermost sediment layers were affected by poor redox cycling. Such conditions imply a limited mixed-layer depth and inefficient nutrient recycling conditioned by hypoxic bottom-waters, allowed by poor circulation and high sedimentation rates in depocenters of the Zliechov Basin. Hypoxic conditions are further supported by (1) dominance of trace-fossils produced by infaunal deposit feeders, (2) high abundance of hypoxiatolerant agglutinated foraminifer Bathysiphon, and (3) high abundance of Chondrites with ~0.5 mm-sized branches. Oxygen-deficient bottom-conditions can thus simultaneously explain the rarity of benthic carbonate-producing macroinvertebrates and high standing abundance of tolerant soft-shell and agglutinated organisms in spotted deposits.

Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine Sertoli cells.

PubMed

Menegazzo, Massimo; Zuccarello, Daniela; Luca, Giovanni; Ferlin, Alberto; Calvitti, Mario; Mancuso, Francesca; Calafiore, Riccardo; Foresta, Carlo

2011-10-01

Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. Fresh sperm cells (5 ml of 20 × 10(6)/ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.

Implications for Late Pleistocene Mastodon Diet from Opal Phytoliths in Tooth Calculus

NASA Astrophysics Data System (ADS)

Gobetz, Katrina E.; Bozarth, Steven R.

2001-03-01

Calculus removed from the molar teeth of four American mastodons (Mammut americanum) contained opal phytoliths which reflect major dietary components. Three samples contained abundant grass phytoliths (ca. 86% of total), with long cells and trapezoidal pooid short cells dominant (ca. 25 and 31%, respectively). Dicot phytoliths from hackberry (Celtis sp.) seeds and indeterminate deciduous trees were rare (1-3%), though well preserved, whereas phytoliths from conifer trees were not recognizable in any of the samples. Comparative analysis of calculus from modern and fossil browsers and mixed feeders implies that dicots and conifers are nearly invisible in the phytolith record. This scarcity may result from poor preservation, low silica production in woody taxa, and/or animals' selection of young, silica-poor leaves and shoots. However, abundant grass phytoliths in the mixed feeders suggest that presence versus absence of grass phytoliths may distinguish mixed feeders and grazers from browsers. Mastodons are traditionally considered browsers, but grass phytolith assemblages in three individual mastodons contained similarly high concentrations of pooids, suggesting that these grasses were a significant part of the diet. Abundant pooid phytoliths, in addition to diatoms, indicate that these mastodons grazed in a cool, moist late Pleistocene environment, possibly near water.

Food web structure and seasonality of slope megafauna in the NW Mediterranean elucidated by stable isotopes: Relationship with available food sources

NASA Astrophysics Data System (ADS)

Papiol, V.; Cartes, J. E.; Fanelli, E.; Rumolo, P.

2013-03-01

The food-web structure and seasonality of the dominant taxa of benthopelagic megafauna (fishes and decapods) on the middle slope of the Catalan Sea (Balearic Basin, NW Mediterranean) were investigated using the carbon and nitrogen stable isotope ratios of 29 species. Macrofauna (infauna, suprabenthos and zooplankton) were also analysed as potential prey. Samples were collected on a seasonal basis from 600 to 1000 m depth between February 2007 and February 2008. The fishes and decapods were classified into feeding groups based on the literature: benthic feeders (including suprabenthos) and zooplankton feeders, the latter further separated into migratory and non-migratory species. Decapods exhibited depleted δ15N and enriched δ13C compared to fishes. Annual mean δ13C of fishes ranged from - 19.15‰ (Arctozenus risso) to - 16.65‰ (Phycis blennoides) and of δ15N from 7.27‰ (Lampanyctus crocodilus) to 11.31‰ (Nezumia aequalis). Annual mean values of δ13C of decapods were from - 18.94‰ (Sergestes arcticus) to - 14.78‰ (Pontophilus norvegicus), and of δ15N from 6.36‰ (Sergia robusta) to 9.72‰ (Paromola cuvieri). Stable isotopes distinguished well amongst the 3 feeding guilds established a priori, pointing to high levels of resource partitioning in deep-sea communities. The trophic structure of the community was a function of the position of predators along the benthic-pelagic gradient, with benthic feeders isotopically enriched relative to pelagic feeders. This difference allowed the identification of two food webs based on pelagic versus benthic consumption. Prey and predator sizes were also important in structuring the community. The most generalised seasonal pattern was δ13C depletion from winter to spring and summer, especially amongst migratory macroplankton feeders. This suggests greater consumption of pelagic prey, likely related with increases in pelagic production or with ontogenic migrations of organisms from mid-water to the Benthic Boundary Layer (BBL). δ15N enrichment was detected in periods of water column stratification, particularly amongst benthic feeder fishes. Megafauna relied on a single source of nutrition after peaks in surface production, presumably marine snow. Conversely, a larger array of food sources, probably from advection, sustained the community in periods of water column stratification. Benthic feeder δ13C values of both taxa were positively correlated with fluorescence measured 5 m above the seabed and negatively correlated with total organic carbon in the sediments, both being food sources for deposit feeding macroinfauna. Macroplankton feeder δ13C values were linked to environmental variables related to vertical transport from surface production, i.e. lipids and chlorophyll and their degradation products, likely due to their stronger reliance on sinking phytodetritus through consumption of planktonic prey.

Effect of mitomycin-C on human foreskin fibroblasts used as feeders in human embryonic stem cells: immunocytochemistry MIB1 score and DNA ploidy and apoptosis evaluated by flow cytometry.

PubMed

Nieto, A; Cabrera, C M; Catalina, P; Cobo, F; Barnie, A; Cortés, J L; Barroso del Jesus, A; Montes, R; Concha, A

2007-03-01

Mitomycin C (MMC) treatment has been used to arrest cell proliferation but not much is known about the effect of MMC on human foreskin fibroblasts (HFF) used as feeders for human embryonic stem cells (hESC). We tested the ability of MMC to stop the proliferation of HFF and to induce apoptosis. MMC inhibited the proliferation of HFF at 10 microg/ml over 2.5h of MMC treatment showing a decrease in the proliferation index measured by Ki-67 and S and G2/M phases related to active HFF. A low percentage of cells showed necrotic or apoptotic features using different lengths of incubation. Over time, the majority of cells remained in a mitotically inactive state. The percentage of apoptotic cells increased from day 2 to day 10, at the same time as the necrotic ones increased. The HS181 hESC line grew in an undifferentiated state on inactive HFF throughout the study.

Unsuccessful derivation of human embryonic stem cell lines from pairs of human blastomeres.

PubMed

Fong, Chui-Yee; Richards, Mark; Bongso, Ariff

2006-08-01

Human embryonic stem cells (hESC) that differentiate into all three primordial germ layers have been established. Differentiation of these cells into desirable lineages offers hope for future transplantation therapies. Currently, hESC lines are derived from the inner cell mass (ICM) of blastocysts, leading to destruction of the embryo, and thus the process is ethically controversial. Successful attempts at deriving hESC lines from blastomeres without destruction of the ensuing embryo have not been reported. One or two blastomeres are routinely biopsied from 8-cell embryos for preimplantation genetic diagnosis. In this study it was therefore attempted to derive hESC lines from paired blastomeres. Of 66 pairs of 8-cell stage blastomeres, four pairs produced two morula and two blastocyst-like structures. When plated on mitomycin-C-treated mouse embryonic fibroblasts, one morula and one blastocyst-like structure separately produced small colonies containing hESC-like cells with prominent nucleoli and high nuclear-cytoplasmic ratios. When these colonies were detached and plated onto fresh feeders, there was no further colony formation or ensuing hESC lines. The results showed that it might not be possible to derive hESC lines directly from paired blastomeres. A minimum number of blastomeres in close contact with one another may be required to successfully generate an hESC line as blastomeres, like ICM and hESC cells, may be 'social' cells.

Novel Methods to Determine Feeder Locational PV Hosting Capacity and PV Impact Signatures

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

Reno, Matthew J.; Coogan, Kyle; Seuss, John

Often PV hosting capacity analysis is performed for a limited number of distribution feeders. For medium - voltage distribution feeders, previous results generally analyze less than 20 feeders, and then the results are extrapolated out to similar types of feeders. Previous hosting capacity research has often focused on determining a single value for the hosting capacity for the entire feeder, whereas this research expands previous hosting capacity work to investigate all the regions of the feeder that may allow many different hosting capacity values wit h an idea called locational hosting capacity (LHC)to determine the largest PV size that canmore » be interconnected at different locations (buses) on the study feeders. This report discusses novel methods for analyzing PV interconnections with advanced simulati on methods. The focus is feeder and location - specific impacts of PV that determine the locational PV hosting capacity. Feeder PV impact signature are used to more precisely determine the local maximum hosting capacity of individual areas of the feeder. T he feeder signature provides improved interconnection screening with certain zones that show the risk of impact to the distribution feeder from PV interconnections.« less

Round-bale feeder design affects hay waste and economics during horse feeding.

PubMed

Martinson, K; Wilson, J; Cleary, K; Lazarus, W; Thomas, W; Hathaway, M

2012-03-01

Many horse owners find round bales convenient, less labor intensive, and more affordable than other hay types, but report an inability to control horse BW gain and excessive hay waste. The objectives were to compare hay waste, hay intake, and payback of 9 round-bale feeders and a no-feeder control when used during horse feeding. Nine round-bale feeders were tested: Cinch Net, Cone, Covered Cradle, Hayhut, Hay Sleigh, Ring, Tombstone, Tombstone Saver, and Waste Less. Each feeder design was placed on the ground in a dirt paddock. Five groups of 5 horses were fed in rotation for a 4-d period with each feeder. Every fourth day, groups were rotated among paddocks and a new round bale was placed in each feeder. In the 5 paddocks used, 5 feeders were installed for d 1 through 20, and the remaining 4 feeders and no-feeder control were installed for d 21 through 40. Groups of horses were sequentially assigned to feeders using two 5 × 5 Latin squares, the first for d 1 through 20, the second for d 21 through 40. Horse groups of similar age, BW, breed, and sex were formed from 25 Quarter Horse and Thoroughbred geldings and open mares (means: 11 yr; 541 kg of BW). Hay on the ground surrounding the feeder was collected daily, dried, and weighed. The total amount of hay removed around each feeder for a 4-d period was considered waste. Dry matter intake was estimated as the difference between hay disappearance and waste. Number of months for the reduction in waste to repay feeder cost (payback) were calculated using hay valued at $110/t, and improved feeder efficiency over the control. Feeder design did not affect hay intake (P > 0.05); all feeders resulted in an estimated hay intake of 2.0 to 2.4% BW; the no-feeder control resulted in a reduced intake of 1.3% BW (P = 0.001). Mean percentage of hay waste differed among feeders (P < 0.001): Waste Less, 5%; Cinch Net, 6%; Hayhut, 9%; Covered Cradle, 11%; Tombstone Saver, 13%; Tombstone, Cone, and Ring, 19%; Hay Sleigh, 33%; and no-feeder control, 57%. Feeder design also affected payback (P < 0.01). The Cinch Net paid for itself in less than 1 mo; Tombstone and Ring, 2 mo; Hayhut and Tombstone Saver, 4 mo; Hay Sleigh, 5 mo; Waste Less, 8 mo; Cone, 9 mo; and Covered Cradle, 20 mo. Round-bale feeder design affected hay waste and payback, but not estimated hay intake or BW change during horse feeding.

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

PubMed

Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.

PubMed

Sharma, Ruchi; George, Aman; Kamble, Nitin Manchindra; Singh, Karn Pratap; Chauhan, Manmohan Singh; Singla, Suresh Kumar; Manik, Radhey Sham; Palta, Prabhat

2011-12-01

A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

Effects of feeder design and changing source of water to a location separate from the wet-dry feeder at 4 or 8 weeks before harvest on growth, feeding behavior, and carcass characteristics of finishing pigs.

PubMed

Bergstrom, J R; Nelssen, J L; Edwards, L N; Tokach, M D; Dritz, S S; Goodband, R D; DeRouchey, J M

2012-12-01

Our objectives were to compare a conventional dry (5-space, 152.4-cm-wide) and a wet-dry (double-sided, each side = 38.1-cm-wide single space) feeder and to determine if changing the source of water to a location separate from a wet-dry feeder would result in improved G:F and carcass characteristics. Water supply to the wet-dry feeder was shut off and the cup waterer was turned on in 8 pens at 8 (d 69) or 4 (d 97) wk prior to harvest. For the remaining 8 wet-dry feeder pens, the feeder provided the sole water source for the entire experiment. A total of 1,296 pigs (PIC, 337 × 1050; initially 19.4 kg BW) were used, with 27 pigs/pen (14 barrows and 13 gilts) and 24 pens/feeder design. From d 0 to 69, pigs fed with the wet-dry feeder had increased (P < 0.05) ADG, ADFI, G:F, and d 69 BW compared with those using the conventional dry feeder. Overall (d 0 to 124), pigs using fed with the water source in the wet-dry feeder the entire time had greater (P < 0.05) ADG, ADFI, final BW, and HCW the other treatments. The overall G:F was not different (P > 0.05) among pigs fed with the different feeder treatments. Pigs fed with the wet-dry feeder where water source was changed at 4 wk before harvest had greater (P < 0.05) ADG than pigs that used a conventional dry feeder. Pigs where the water source was changed at 4 wk had greater (P < 0.05) ADFI than those were the water source was changed 8 wk prior to harvest, and for pigs fed with the conventional dry feeder ADFI was intermediate. Back fat depth of pigs where the water source was changed at 8 wk before harvest was reduced (P < 0.05) compared with all other treatments and LM depth was greater (P < 0.05) than that of pigs using a conventional dry feeder and where the water source was changed at 4 week before harvest. Pigs fed using the wet-dry feeder visited the feeder less frequently (P < 0.05) and spent less total time at the feeder (P < 0.05) than those fed with the conventional dry feeder. The differences in feeding patterns remained even after the access to water was removed from the wet-dry feeder, with no change in the amount of aggressive behavior observed at the feeder. Pigs fed with a wet-dry feeder had an increased growth rate compared with those fed with a conventional dry feeder. Although measures of carcass leanness were improved by changing the location of the water, removing the water from the feeder also eliminated any net improvement in BW from using a wet-dry feeder.

95. VIEW OF ZINC FEEDER FROM SOUTHEAST. NOTE FEEDER CONE ...

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

95. VIEW OF ZINC FEEDER FROM SOUTHEAST. NOTE FEEDER CONE AND PIPING FROM VACUUM RECEIVER ON LEFT. PRECIPITATE PUMP MOTOR MOUNT VISIBLE BELOW FEEDER STAIRS, PUMP AND MOTOR MISSING. SUMPS ARE LOCATED UNDER THIS FLOOR, WITH ACCESS TO HATCH TO THE RIGHT OF FEEDER STAIR. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

Hygiene at winter bird feeders in a southwestern Ontario city.

PubMed Central

Prescott, J F; Hunter, D B; Campbell, G D

2000-01-01

To further understand the source of the epidemic of salmonellosis in some species of birds using bird feeders in southern Ontario in the winter of 1997-1998, 124 bird feeder stations were examined for their state of hygiene and for Salmonella on 5 occasions during the winter of 1999 in a city of 100,000 people in southwestern Ontario. No Salmonella were isolated from feed contaminated with feces recovered from the feeders. Squirrel-proof feeders were significantly less contaminated with feces than were other feeder types (hopper, platform, silo), which did not differ significantly in their hygiene scores. Contamination of squirrel-proof feeders increased significantly through the course of the study, but other feeder types showed no significant change. Hygiene was poorer if feeders were maintained equally by both male and female household members, particularly as they grew older, but no age or gender effect was observed if only one person was largely responsible for maintaining the feeders. We concluded that winter bird feeder stations in a southern Ontario city were not contaminated with Salmonella but that bird feeder stations could be designed better to reduce fecal contamination of feed. PMID:10992987

Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis.

PubMed

Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

2013-09-06

Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy. Copyright © 2013 Elsevier Inc. All rights reserved.

The synergistic effect of beta-boswellic acid and Nurr1 overexpression on dopaminergic programming of antioxidant glutathione peroxidase-1-expressing murine embryonic stem cells.

PubMed

Abasi, M; Massumi, M; Riazi, G; Amini, H

2012-10-11

Parkinson's disease (PD) is a neurodegenerative disorder in which the nigro-striatal dopaminergic (DAergic) neurons have been selectively lost. Due to side effects of levodopa, a dopamine precursor drug, recently cell replacement therapy for PD has been considered. Lack of sufficient amounts of, embryos and ethical problems regarding the use of dopamine-rich embryonic neural cells have limited the application of these cells for PD cell therapy. Therefore, many investigators have focused on using the pluripotent stem cells to generate DAergic neurons. This study is aimed first to establish a mouse embryonic stem (mES) cell line that can stably co-express Nurr1 (Nuclear receptor subfamily 4, group A, member 2) transcription factor in order to efficiently generate DAergic neurons, and glutathione peroxidase-1 (GPX-1) to protect the differentiated DAergic-like cells against oxidative stress. In addition to genetic engineering of ES cells, the effect of Beta-boswellic acid (BBA) on DAergic differentiation course of mES cells was sought in the present study. To that end, the feeder-independent CGR8 mouse embryonic stem cells were transduced by Nurr1- and GPX-1-harboring Lentiviruses and the generated Nurr1/GPX-1-expresssing ES clones were characterized and verified. Gene expression analyses demonstrated that BBA treatment and overexpression of Nurr1 has a synergistic effect on derivation of DAergic neurons from Nurr1/GPX-1-expressing ES cells. The differentiated cells could exclusively synthesize and secrete dopamine in response to stimuli. Overexpression of GPX-1 in genetically engineered Nurr1/GPX-1-ES cells increased the viability of these cells during their differentiation into CNS stem cells. In conclusion, the results demonstrated that Nurr1-overexpressing feeder-independent ES cells like the feeder-dependent ES cells, can be efficiently programmed into functional DAergic neurons and additional treatment of cells by BBA can even augment this efficiency. GPX-1 overexpression in Nurr1/GPX-1-ES cells increases the viability of differentiated CNS stem-like cells. The result of this study may have impact on future stem cell therapy of PD. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Basic FGF Support of Human Embryonic Stem Cell Self-Renewal

PubMed Central

Levenstein, Mark E.; Ludwig, Tenneille E.; Xu, Ren-He; Llanas, Rachel A.; VanDenHeuvel-Kramer, Kaitlyn; Manning, Daisy; Thomson, James A.

2015-01-01

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic FGF (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. Recently, it has been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Here we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100 and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture the cells formed teratomas when injected into SCID-beige mice, and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells, and suggest that fibroblasts and fibroblast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold. PMID:16282444

Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.

PubMed Central

Sutherland, H J; Lansdorp, P M; Henkelman, D H; Eaves, A C; Eaves, C J

1990-01-01

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells. Images PMID:2333304

Evaluation of ERDA-sponsored coal feed system development

NASA Technical Reports Server (NTRS)

Phen, R. L.; Luckow, W. K.; Mattson, L.; Otth, D.; Tsou, P.

1977-01-01

Coal feeders were evaluated based upon criteria such as technical feasibility, performance (i.e. ability to meet process requirements), projected life cycle costs, and projected development cost. An initial set of feeders was selected based on the feeders' cost savings potential compared with baseline lockhopper systems. Additional feeders were considered for selection based on: (1) increasing the probability of successful feeder development; (2) application to specific processes; and (3) technical merit. A coal feeder development program is outlined.

Development and evaluation of a new composite Laserskin graft.

PubMed

Lam, P K; Chan, E S; To, E W; Lau, C H; Yen, S C; King, W W

1999-11-01

Tremendous effort has been made to improve the graft take rate of cultured epidermal autograph. The purpose of this study is to develop and evaluate a new composite Laserskin graft (CLSG) as a human skin substitute for wound resurfacing. The seeding efficacy of cultured keratinocytes on plain Laserskin was compared with the 3T3 cell-seeded Laserskin and allogenic fibroblast-populated Laserskin. Three different types of CLSG, 2 cm in diameter each, were prepared and tested in rats. Type A CLSG consisted of proliferative allogenic rat fibroblasts on both sides of the Laserskin with autologous keratinocytes also on the upper side. Fibroblasts and keratinocytes were seeded only on the upper side of the Laserskin in type B CLSG. Keratinocytes alone were seeded on plain Laserskin in type C CLSG. Type B CLSG consisting of autologous keratinocytes and autologous dermal fibroblasts was tested on five selected wounds (5x5 cm each) of a patient with full-thickness burn. In another burn patient, type B CLSG consisting of autologous keratinocytes and allogenic dermal fibroblasts was grafted onto three wounds (5x5 cm each). The seeding efficacy of human keratinocytes on plain Laserskin increased from 75% to 95% when proliferative allogenic fibroblasts were grown as a feeder layer on the Laserskin. The seeding efficacy of rat keratinocytes increased from 36% to 88% in the presence of a proliferative allogenic fibroblast feeder layer, whereas human/rat keratinocytes had respective seeding efficacy of 98%/91% on Laserskin preseeded with mitomycin C-treated 3T3 cells. Skin biopsies of grafted type A CLSG on day 14 after grafting showed complete epithelialization without severe inflammation in 16 of 20 (80%) grafted surgical wounds in rats. There were eight (40%) and seven (35%) "takes" of the CLSG in types B and C, respectively. The infection rate in type B CLSG was two (10%). There was one (5%) infection in types A and C. The respective take rates on the two patients grafted with type B CLSG were 60% and 100%. The animal experiment and the preliminary clinical data showed that the CSLGs consisting of autologous keratinocytes and of autologous/allogenic fibroblasts are good human skin substitutes in terms of durability, biocompatibility, high seeding efficacy for keratinocytes, high graft take rate, and low infection rate.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

PubMed

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro.

PubMed

Ahn, Yong-Oon; Kim, Saerom; Kim, Tae Min; Song, Eun Young; Park, Myoung Hee; Heo, Dae Seog

2013-09-01

Adoptive cell transfer of ex vivo-activated natural killer (NK) cells is a promising therapy for cancer treatment. Because of inhibitory signaling through killer immunoglobulin-like receptor (KIR)-KIR ligands, KIR-mismatched allogeneic NK cell transfer is considered to be a more effective strategy than is autologous transfer. However, purified NK cells do not expand well enough in vitro with good manufacturing practice-compliant components for clinical use. Some investigators have developed selective expansion of NK cells from peripheral blood mononuclear cells, but these cells have the risk of graft-versus-host disease in allogeneic settings because of T cells contamination. In this study, we developed a novel method for NK cell activation and expansion. Using only good manufacturing practice-compliant components and autologous feeder cells, once purified NK cells were effectively expanded (2500-fold at day 17). The expanded cells were highly purified NK cells, and the use of these cells is suitable for allogeneic transfer without the risk of graft-versus-host disease induction. Importantly, the expanded NK cells also showed enhanced cytotoxicity compared with NK cells conventionally expanded by recombinant human interleukin 2. Finally, induction of NKG2D ligand expression on feeder cells implies that the NKG2D-NKG2DL interaction may play a role in NK cell expansion. In conclusion, this method can be used to obtain NK cells for more successful allogeneic NK cell adoptive transfer for use in antitumor immune therapy.

Colour preferences of UK garden birds at supplementary seed feeders.

PubMed

Rothery, Luke; Scott, Graham W; Morrell, Lesley J

2017-01-01

Supplementary feeding of garden birds generally has benefits for both bird populations and human wellbeing. Birds have excellent colour vision, and show preferences for food items of particular colours, but research into colour preferences associated with artificial feeders is limited to hummingbirds. Here, we investigated the colour preferences of common UK garden birds foraging at seed-dispensing artificial feeders containing identical food. We presented birds simultaneously with an array of eight differently coloured feeders, and recorded the number of visits made to each colour over 370 30-minute observation periods in the winter of 2014/15. In addition, we surveyed visitors to a garden centre and science festival to determine the colour preferences of likely purchasers of seed feeders. Our results suggest that silver and green feeders were visited by higher numbers of individuals of several common garden bird species, while red and yellow feeders received fewer visits. In contrast, people preferred red, yellow, blue and green feeders. We suggest that green feeders may be simultaneously marketable and attractive to foraging birds.

Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

PubMed Central

2013-01-01

Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

Precision powder feeder

DOEpatents

Schlienger, M. Eric; Schmale, David T.; Oliver, Michael S.

2001-07-10

A new class of precision powder feeders is disclosed. These feeders provide a precision flow of a wide range of powdered materials, while remaining robust against jamming or damage. These feeders can be precisely controlled by feedback mechanisms.

Isolation and culture of rabbit embryonic stem cells.

PubMed

Honda, Arata

2013-01-01

Mammalian stem cells are invaluable research resources for the study of cell and embryonic development as well as practical tools for use in the production of genetically engineered animals and further therapeutics. It is important that we further our knowledge and understanding of a variety of stem cells from several different animal species before trials in humans commence. Here we describe methods for establishing rabbit embryonic stem (rES) cell lines with indefinite proliferation potential. rES cells attain maximum proliferation potential when cultured at a feeder cell density of one-sixth of that of full confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Fibroblast growth factor (FGF)2 can maintain the undifferentiated status of rES cells; however leukemia inhibitory factor (LIF) is dispensable. Under optimized conditions, rES cells could be passaged by trypsinization 50 times. This culture system enabled efficient gene transduction and clonal expansion from single cells. rES cells grew as flat monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo, respectively. Characterization of ES cells from different species is important for establishing common features of pluripotency. We have demonstrated the similarity of ES cells between rabbit and humans. These cell lines could be applied directly using gene-targeting techniques, or in combination with induced pluripotent stem cells. Thus, rES cells are a suitable model for studying human transplantation therapy and disease treatments.

Meta-analysis on the effects of the physical environment, animal traits, feeder and feed characteristics on the feeding behaviour and performance of growing-finishing pigs.

PubMed

Averós, X; Brossard, L; Dourmad, J Y; de Greef, K H; Edwards, S A; Meunier-Salaün, M C

2012-08-01

A meta-analysis, using information from 45 experiments on growing-finishing pigs published in 39 manuscripts, was carried out to determine the simultaneous effects of the physical environment (space allowance, group size, flooring conditions, temperature, presence of enrichment), pig traits (initial body weight (BW) for each studied time interval, sex, genetics), feeder characteristics (water provision within the feeder, feeder design (individual/collective), feeder places/pig, presence of feeder protection) and feed characteristics (feed allowance (ad libitum/restricted), net energy content, crude protein (CP) content), as well as their potential interactions, on the feeding behaviour and performance of growing-finishing pigs. The detrimental effect of low temperature on performance was particularly evident for restricted-fed pigs (P < 0.05). At reduced feeder space allowance, a reduction in the percentage of time spent eating was predicted when increasing initial BW, whereas the opposite was predicted for larger feeder space allowances (P < 0.001). The reduction in visit duration to the feeder in higher BW groups became gradually more important with increasing feeder space allowance (P < 0.01), whereas the increase in the ingestion rate and average daily feed intake (ADFI) with increasing initial BW became smaller with increasing feeder space (P < 0.05). The model predicted a reduction in feed conversion ratio (FCR) with increasing group size (P < 0.05) and floor space allowance (P < 0.01) and on solid floors with or without bedding (P < 0.05). In comparison with other feeders, wet/dry feeders were associated with more frequent but shorter feeder visits (P < 0.05), higher ingestion rates (P < 0.001) and higher ADFI (P < 0.10). The use of protection within individual feeders increased the time spent feeding (P < 0.001), reduced the number of visits per day (P < 0.01), the ingestion rate (P < 0.001) and FCR (P < 0.01) in comparison with other feeder types. Sex modulated the effect of the number of feeder places/pig on FCR (P < 0.05), with a gradual reduction of FCR in entire males and females when increasing feeder space allowance. Genetics tended to modulate the effect of diets' CP content on FCR (P < 0.10). Overall, these results may contribute to the improvement of the welfare and performance of growing-finishing pigs by a better knowledge of the influence of the rearing environment and may help optimize the feeding strategies in current production systems.

Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

PubMed

Catalina, Puri; Montes, Rosa; Ligero, Gertru; Sanchez, Laura; de la Cueva, Teresa; Bueno, Clara; Leone, Paola E; Menendez, Pablo

2008-10-03

The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare...

30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare...

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Improved Small-Particle Powders for Plasma Spraying

NASA Technical Reports Server (NTRS)

Nguyen, QuynhGiao, N.; Miller, Robert A.; Leissler, George W.

2005-01-01

Improved small-particle powders and powder-processing conditions have been developed for use in plasma spray deposition of thermal-barrier and environmental barrier coatings. Heretofore, plasma-sprayed coatings have typically ranged in thickness from 125 to 1,800 micrometers. As explained below, the improved powders make it possible to ensure complete coverage of substrates at unprecedently small thicknesses of the order of 25 micrometers. Plasma spraying involves feeding a powder into a hot, high-velocity plasma jet. The individual powder particles melt in the plasma jet as they are propelled towards a substrate, upon which they splat to build up a coating. In some cases, multiple coating layers are required. The size range of the powder particles necessarily dictates the minimum thickness of a coating layer needed to obtain uniform or complete coverage. Heretofore, powder particle sizes have typically ranged from 40 to 70 micrometers; as a result, the minimum thickness of a coating layer for complete coverage has been about 75 micrometers. In some applications, thinner coatings or thinner coating layers are desirable. In principle, one can reduce the minimum complete-coverage thickness of a layer by using smaller powder particles. However, until now, when powder particle sizes have been reduced, the powders have exhibited a tendency to cake, clogging powder feeder mechanisms and feed lines. Hence, the main problem is one of synthesizing smaller-particle powders having desirable flow properties. The problem is solved by use of a process that begins with a spray-drying subprocess to produce spherical powder particles having diameters of less than 30 micrometers. (Spherical-particle powders have the best flow properties.) The powder is then passed several times through a commercial sifter with a mesh to separate particles having diameters less than 15 micrometers. The resulting fine, flowable powder is passed through a commercial fluidized bed powder feeder into a plasma spray jet.

Feeder density enhances house finch disease transmission in experimental epidemics.

PubMed

Moyers, Sahnzi C; Adelman, James S; Farine, Damien R; Thomason, Courtney A; Hawley, Dana M

2018-05-05

Anthropogenic food provisioning of wildlife can alter the frequency of contacts among hosts and between hosts and environmental sources of pathogens. Despite the popularity of garden bird feeding, few studies have addressed how feeders influence host contact rates and disease dynamics. We experimentally manipulated feeder density in replicate aviaries containing captive, pathogen-naive, groups of house finches ( Haemorhous mexicanus ) and continuously tracked behaviours at feeders using radio-frequency identification devices. We then inoculated one bird per group with Mycoplasma gallisepticum (Mg), a common bacterial pathogen for which feeders are fomites of transmission, and assessed effects of feeder density on house finch behaviour and pathogen transmission. We found that pathogen transmission was significantly higher in groups with the highest density of bird feeders, despite a significantly lower rate of intraspecific aggressive interactions relative to the low feeder density groups. Conversely, among naive group members that never showed signs of disease, we saw significantly higher concentrations of Mg-specific antibodies in low feeder density groups, suggesting that birds in low feeder density treatments had exposure to subclinical doses of Mg. We discuss ways in which the density of garden bird feeders could play an important role in mediating the intensity of Mg epidemics.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'. © 2018 The Author(s).

Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions.

PubMed

Kanke, Kosuke; Masaki, Hideki; Saito, Taku; Komiyama, Yuske; Hojo, Hironori; Nakauchi, Hiromitsu; Lichtler, Alexander C; Takato, Tsuyoshi; Chung, Ung-Il; Ohba, Shinsuke

2014-06-03

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts.

PubMed

Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming Liu; Wang, Yujiong; He, Yulong

2017-06-01

Induced pluripotent stem cells (iPS) represent an important tool to develop disease‑modeling assays, drug testing assays and cell‑based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer‑binding transcription factor 4 (Oct4), sex determining region Y‑box 2 (Sox2), Kruppel‑like factor 4 and c‑Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder‑free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin‑coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases.

Colour preferences of UK garden birds at supplementary seed feeders

PubMed Central

Rothery, Luke; Scott, Graham W.

2017-01-01

Supplementary feeding of garden birds generally has benefits for both bird populations and human wellbeing. Birds have excellent colour vision, and show preferences for food items of particular colours, but research into colour preferences associated with artificial feeders is limited to hummingbirds. Here, we investigated the colour preferences of common UK garden birds foraging at seed-dispensing artificial feeders containing identical food. We presented birds simultaneously with an array of eight differently coloured feeders, and recorded the number of visits made to each colour over 370 30-minute observation periods in the winter of 2014/15. In addition, we surveyed visitors to a garden centre and science festival to determine the colour preferences of likely purchasers of seed feeders. Our results suggest that silver and green feeders were visited by higher numbers of individuals of several common garden bird species, while red and yellow feeders received fewer visits. In contrast, people preferred red, yellow, blue and green feeders. We suggest that green feeders may be simultaneously marketable and attractive to foraging birds. PMID:28212435

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

PubMed

Liu, Weiqiang; Yin, Yifei; Long, Xiaolin; Luo, Yumei; Jiang, Yonghua; Zhang, Wenhong; Du, Hongzi; Li, Shaoying; Zheng, Yuhong; Li, Qing; Chen, Xinjie; Liao, Baoping; Xiao, Guohong; Wang, Weihua; Sun, Xiaofang

2009-04-01

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

Egr-1: A Candidate Transcription Factor Involved in Molecular Processes Underlying Time-Memory.

PubMed

Shah, Aridni; Jain, Rikesh; Brockmann, Axel

2018-01-01

In honey bees, continuous foraging is accompanied by a sustained up-regulation of the immediate early gene Egr-1 (early growth response protein-1) and candidate downstream genes involved in learning and memory. Here, we present a series of feeder training experiments indicating that Egr-1 expression is highly correlated with the time and duration of training even in the absence of the food reward. Foragers that were trained to visit a feeder over the whole day and then collected on a day without food presentation showed Egr-1 up-regulation over the whole day with a peak expression around 14:00. When exposed to a time-restricted feeder presentation, either 2 h in the morning or 2 h in the evening, Egr-1 expression in the brain was up-regulated only during the hours of training. Foragers that visited a feeder in the morning as well as in the evening showed two peaks of Egr-1 expression. Finally, when we prevented time-trained foragers from leaving the colony using artificial rain, Egr-1 expression in the brains was still slightly but significantly up-regulated around the time of feeder training. In situ hybridization studies showed that active foraging and time-training induced Egr-1 up-regulation occurred in the same brain areas, preferentially the small Kenyon cells of the mushroom bodies and the antennal and optic lobes. Based on these findings we propose that foraging induced Egr-1 expression can get regulated by the circadian clock after time-training over several days and Egr-1 is a candidate transcription factor involved in molecular processes underlying time-memory.

Feed intake and behavior of dairy goats when offered an elevated feed bunk.

PubMed

Neave, Heather W; von Keyserlingk, Marina A G; Weary, Daniel M; Zobel, Gosia

2018-04-01

Goats are browsers and select vegetation at various heights when foraging. On commercial farms, dairy goats are typically fed from low-level feed bunks. The objective of this study was to determine how feed intake and feeding behavior vary when goats are offered feed at variable heights, with the potential of evaluating the benefits of offering an elevated feeder to dairy goats. Thirteen Saanen X dairy goats were housed in a home pen with a lying area of wood shavings, where they were pre-exposed for 24 d to 3 feeder heights designed to result in differences in head height while feeding: floor level (head lowered relative to body), head level (head level relative to body), and elevated level (head and neck angled upward). Nine groups of 3 goats each were randomly selected and housed for 24 h in a test pen identical to the home pen except that it contained 1 of each of the 3 feeder heights. Each feeder contained ad libitum chopped alfalfa silage and a top-dressed corn-based supplement, refreshed twice daily. Refusals from inside and under each feeder were weighed to calculate intake. Feed intake increased with increasing feeder height (mean ± SE; 0.18, 0.29, and 0.34 ± 0.04 kg of DM/goat for floor-level, head-level, and elevated-level feeders, respectively). Total feeding time did not vary with feeder height, but feeding rate tended to be faster at the elevated-level feeder (14.5 ± 2.1 g of DM/min) compared with head-level (9.2 ± 2.3 g of DM/min) and floor-level (8.9 ± 2.1 g of DM/min) feeders. Goats visited the floor-level feeder (36.4 ± 8.4 visits/goat) less than the head-level (79.4 ± 8.4 visits/goat) and elevated-level (74.8 ± 8.4 visits/goat) feeders. The number of displacements per minute of feeding time (physical removal of another goat from the feeding place) was greater at the elevated-level feeder (0.46 ± 0.06 displacements/min) compared with the floor-level feeder (0.23 ± 0.06 displacements/min) and tended to differ from the head-level feeder (0.27 ± 0.06 displacements/min). We conclude that goats eat more from an elevated feeder and compete more to access this feeder. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Thermal imaging for assessment of electron-beam freeform fabrication (EBF3) additive manufacturing deposits

NASA Astrophysics Data System (ADS)

Zalameda, Joseph N.; Burke, Eric R.; Hafley, Robert A.; Taminger, Karen M.; Domack, Christopher S.; Brewer, Amy; Martin, Richard E.

2013-05-01

Additive manufacturing is a rapidly growing field where 3-dimensional parts can be produced layer by layer. NASA's electron beam freeform fabrication (EBF3) technology is being evaluated to manufacture metallic parts in a space environment. The benefits of EBF3 technology are weight savings to support space missions, rapid prototyping in a zero gravity environment, and improved vehicle readiness. The EBF3 system is composed of 3 main components: electron beam gun, multi-axis position system, and metallic wire feeder. The electron beam is used to melt the wire and the multi-axis positioning system is used to build the part layer by layer. To insure a quality deposit, a near infrared (NIR) camera is used to image the melt pool and solidification areas. This paper describes the calibration and application of a NIR camera for temperature measurement. In addition, image processing techniques are presented for deposit assessment metrics.

Progression from productive infection to integration and oncogenic transformation in human papillomavirus type 59-immortalized foreskin keratinocytes.

PubMed

Spartz, Helena; Lehr, Elizabeth; Zhang, Benyue; Roman, Ann; Brown, Darron R

2005-05-25

Studies of changes in the virus and host cell upon progression from human papillomavirus (HPV) episomal infection to integration are critical to understanding HPV-related malignant transformation. However, there exist only a few in vitro models of both productive HPV infection and neoplastic progression on the same host background. We recently described a unique foreskin keratinocyte cell line (ERIN 59) that contains HPV 59 (a close relative of HPV 18). Early passages of ERIN 59 cells (passages 9-13) contained approximately 50 copies of episomes/cell, were feeder cell-dependent, and could be induced to differentiate and produce infectious virus in a simple culture system. We now report that late passage cells (passages greater than 50) were morphologically different from early passage cells, were feeder cell independent, and did not differentiate or produce virus. These late passage cells contained HPV in an integrated form. An integration-derived oncogene transcript was expressed in late passage cells. The E2 open reading frame was interrupted in this transcript at nucleotide 3351. Despite a lower viral genome copy number in late passage ERIN 59 cells, expression of E6/E7 oncogene transcripts was similar to early passage cells. We conclude that ERIN 59 cells are a valuable cell line representing a model of progression from HPV 59 episomal infection and virus production to HPV 59 integration and associated oncogenic transformation on the same host background.

35. INTERIOR VIEW, SAME AS ABOVE WITH THE FEEDER ROD ...

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

35. INTERIOR VIEW, SAME AS ABOVE WITH THE FEEDER ROD INSERTED INTO THE MACHINE; NOTE THE WOOD FEEDER ROD AND FEEDER ROD REST ON THE BACK OF THE STOOL - LaBelle Iron Works, Thirtieth & Wood Streets, Wheeling, Ohio County, WV

Long-term Culture and Cloning of Primary Human Bronchial Basal Cells that Maintain Multipotent Differentiation Capacity and CFTR Channel Function.

PubMed

Peters-Hall, Jennifer Ruth; Coquelin, Melissa L; Torres, Michael J; LaRanger, Ryan; Alabi, Busola Ruth; Sho, Sei; Calva-Moreno, Jose Francisco; Thomas, Philip J; Shay, Jerry William

2018-05-03

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome editing tools. Recently, conditional reprogramming of cells (CRC) with a ROCK inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the lifespan of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial growth medium instead of F-medium and 2% oxygen instead of 21% oxygen, that extend HBEC lifespan while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof of concept, CRISPR/Cas9 genome editing and cloning was used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Analytic Considerations and Design Basis for the IEEE Distribution Test Feeders

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

Schneider, K. P.; Mather, B. A.; Pal, B. C.

For nearly 20 years the Test Feeder Working Group of the Distribution System Analysis Subcommittee has been developing openly available distribution test feeders for use by researchers. The purpose of these test feeders is to provide models of distribution systems that reflect the wide diversity in design and their various analytic challenges. Because of their utility and accessibility, the test feeders have been used for a wide range of research, some of which has been outside the original scope of intended uses. This paper provides an overview of the existing distribution feeder models and clarifies the specific analytic challenges thatmore » they were originally designed to examine. Additionally, the paper will provide guidance on which feeders are best suited for various types of analysis. The purpose of this paper is to provide the original intent of the Working Group and to provide the information necessary so that researchers may make an informed decision on which of the test feeders are most appropriate for their work.« less

Analytic Considerations and Design Basis for the IEEE Distribution Test Feeders

DOE PAGES

Schneider, K. P.; Mather, B. A.; Pal, B. C.; ...

2017-10-10

For nearly 20 years the Test Feeder Working Group of the Distribution System Analysis Subcommittee has been developing openly available distribution test feeders for use by researchers. The purpose of these test feeders is to provide models of distribution systems that reflect the wide diversity in design and their various analytic challenges. Because of their utility and accessibility, the test feeders have been used for a wide range of research, some of which has been outside the original scope of intended uses. This paper provides an overview of the existing distribution feeder models and clarifies the specific analytic challenges thatmore » they were originally designed to examine. Additionally, the paper will provide guidance on which feeders are best suited for various types of analysis. The purpose of this paper is to provide the original intent of the Working Group and to provide the information necessary so that researchers may make an informed decision on which of the test feeders are most appropriate for their work.« less

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

PubMed

Burton, Peter; Adams, David R; Abraham, Achamma; Allcock, Robert W; Jiang, Zhong; McCahill, Angela; Gilmour, Jane; McAbney, John; Kaupisch, Alexandra; Kane, Nicole M; Baillie, George S; Baker, Andrew H; Milligan, Graeme; Houslay, Miles D; Mountford, Joanne C

2010-12-15

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Effect of artificial feeders on pollen loads of the hummingbirds of Cerro de la Muerte, Costa Rica.

PubMed

Avalos, Gerardo; Soto, Alejandra; Alfaro, Willy

2012-03-01

Although sugar-water feeders are commonly used by enthusiasts to attract hummingbirds, little is known about how they affect hummingbird behavior and flower use. We studied the highland hummingbird assemblage of Cerro de La Muerte, Costa Rica, both at a site with permanent feeders (La Georgina Restaurant) and further from it. We examined how feeder use and monopolization affected seasonal changes in pollen loads during four sampling periods, including dry and wet seasons, from 2003-2005. We expected that species monopolizing the feeders would carry little or no pollen whatsoever, and would have pollen loads characterized by low floral diversity, in contrast with species less dependent on feeders. We obtained pollen samples from 183 individuals of four hummingbird species captured around the feeders using mist nets, which were compared with a pollen reference collection of plants with a pollination syndrome by hummingbirds. The same methods were implemented at a site 3km away from the feeders. Feeder usage was quantified by counting the number of times hummingbirds drank from the feeders in periods of 4min separated by 1min. The effects of hummingbird species and season on pollen load categories were assessed using a nominal logistic regression. The alpha species at the site, the Fiery-throated Hummingbird (Panterpe insignis), dominated the feeders during the dry season. Meanwhile, in the wet season, feeder usage was more evenly distributed across species, with the exception of the Volcano Hummingbird, Selasphorus flammula, which occupies the last place in the dominance hierarchy. Pollen loads of hummingbirds captured near feeders were low in abundance (more than 50% of captured individuals had zero or low pollen loads), and low in species richness (96% of the hummingbirds with pollen from only one plant genus, Centropogon). Overall pollen loads increased during the dry season coinciding with peaks in flower availability, although the majority of captured hummingbirds carried no pollen. Mist nets located 3km from La Georgina returned few captures (one-to-three specimens) per sampling date, contrasting with observations made before feeders were present. These results suggest that sugar-water feeders gather hummingbirds in over considerable distances drawing them away from flowers. The competitive and antagonistic pattern shown between feeders and flowers indicate that natural pollination system could be significantly altered. Supplementing hummingbirds with food seems likely to interfere with pollination networks already stressed by many anthropogenic effects.

A Feeder-Bus Dispatch Planning Model for Emergency Evacuation in Urban Rail Transit Corridors

PubMed Central

Wang, Yun; Yan, Xuedong; Zhou, Yu; Zhang, Wenyi

2016-01-01

The mobility of modern metropolises strongly relies on urban rail transit (URT) systems, and such a heavy dependence causes that even minor service interruptions would make the URT systems unsustainable. This study aims at optimally dispatching the ground feeder-bus to coordinate with the urban rails’ operation for eliminating the effect of unexpected service interruptions in URT corridors. A feeder-bus dispatch planning model was proposed for the collaborative optimization of URT and feeder-bus cooperation under emergency situations and minimizing the total evacuation cost of the feeder-buses. To solve the model, a concept of dummy feeder-bus system is proposed to transform the non-linear model into traditional linear programming (ILP) model, i.e., traditional transportation problem. The case study of Line #2 of Nanjing URT in China was adopted to illustrate the model application and sensitivity analyses of the key variables. The modeling results show that as the evacuation time window increases, the total evacuation cost as well as the number of dispatched feeder-buses decrease, and the dispatched feeder-buses need operate for more times along the feeder-bus line. The number of dispatched feeder-buses does not show an obvious change with the increase of parking spot capacity and time window, indicating that simply increasing the parking spot capacity would cause huge waste for the emergent bus utilization. When the unbalanced evacuation demand exists between stations, the more feeder-buses are needed. The method of this study will contribute to improving transportation emergency management and resource allocation for URT systems. PMID:27676179

Pure cultures and characterization of yak Sertoli cells.

PubMed

Zhang, Hua; Liu, Ben; Qiu, Yuan; Fan, Jiang feng; Yu, Si jiu

2013-12-01

The culture of primary Sertoli cells has become an important resource in the study of their function. However, their use is limited because of contamination of isolated cells with other testicular cells, mainly germ cells. The aim was to establish technique to obtain pure yak Sertoli cells as well as to study the growth kinetics and biological characteristics of Sertoli cells in vitro. Two-step enzyme digestion was used to separate and culture yak Sertoli cells. Cultured using starvation method and the hypotonic treatment were also invented to get pure yak Sertoli cells. Furthermore, the purification of Yak Sertoli cells were identified according to their characteristics, such as bipolar corpuscular around the nucleus and expression of Fasl, in addition to their morphology. The average viability of the Sertoli cells was 97% before freezing and 94.5% after thawing, indicating that cryopreservation in liquid nitrogen had little influence on the viability of Sertoli cells. The growth tendency of yak Sertoli cells was similar to an S-shaped growth curve. Purified yak Sertoli cells frequently exhibited bipolar corpuscula in nucleus after Feulgen staining, and did have a positive reaction of Fasl by the immunocytochemical identification. After recovery chromosomal analysis of Sertoli cells had a normal chromosomal number of 60, comprising 29 pairs of autosomes and one pair of sex chromosomes. Assays for bacteria, fungi and mycoplasmas were negative. In conclusion, yak Sertoli cells have been successfully purified and cultured in vitro, and maintain stable biological characteristics after thawing. Therefore, it will not only preserve the genetic resources of yaks at the cellular level, but also provide valuable materials for transgenic research and feeder layer and nuclear donor cells in yak somatic cell cloning technology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

PubMed

Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

2009-09-01

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

Analysis of material particle motion and optimizing parameters of vibration of two-mass GZS vibratory feeder

NASA Astrophysics Data System (ADS)

Nguyen, Van Xo; Golikov, N. S.

2018-05-01

The structure and kinematics of the two-mass GZS vibratory feeder operation are considered. It is established that the movement of the material's particles on the feeder surface determines its capacity. The development and analysis of the mathematical model of material's particle movement on the two-mass GZS vibratory feeder surface are shown. The results of Matlab optimization of material particles velocity function are given that allows setting rational kinematics of the feeder.

Determining the benefits of Vorticella cell body motion

NASA Astrophysics Data System (ADS)

Specht, Matty C.; Pepper, Rachel E.

2016-11-01

Microscopic sessile suspension feeders are single-celled organisms found in aquatic ecosystems. They live attached to underwater surfaces and create a fluid flow in order to feed on bacteria and debris. They participate in the natural degradation of contaminants in water. Understanding the fluid flow they create enhances our knowledge of their environmental impact. One type of suspension feeder, Vorticella, have been observed to vary their cell body orientation with respect to their surface, but the benefits of this motion are still unknown. We use simulations to investigate the effect of Vorticella body motion on the feeding current and the nutrient flux to the cell body to determine whether or not the motion increases nutrient consumption. We determine the nutrient flux using COMSOL Multiphysics software to solve the advection-diffusion equation with the flow given by a stokeslet model. We use a range of motions similar and dissimilar to that of live Vorticella. We find that most patterns of motion do not increase the nutrient flux, since the Vorticella feed from regions where they already have depleted the water of nutrients. However, it is possible that their motion could help the Vorticella find nutrients that are inhomogenously distributed in water.

ITER Magnet Feeder: Design, Manufacturing and Integration

NASA Astrophysics Data System (ADS)

CHEN, Yonghua; ILIN, Y.; M., SU; C., NICHOLAS; BAUER, P.; JAROMIR, F.; LU, Kun; CHENG, Yong; SONG, Yuntao; LIU, Chen; HUANG, Xiongyi; ZHOU, Tingzhi; SHEN, Guang; WANG, Zhongwei; FENG, Hansheng; SHEN, Junsong

2015-03-01

The International Thermonuclear Experimental Reactor (ITER) feeder procurement is now well underway. The feeder design has been improved by the feeder teams at the ITER Organization (IO) and the Institute of Plasma Physics, Chinese Academy of Sciences (ASIPP) in the last 2 years along with analyses and qualification activities. The feeder design is being progressively finalized. In addition, the preparation of qualification and manufacturing are well scheduled at ASIPP. This paper mainly presents the design, the overview of manufacturing and the status of integration on the ITER magnet feeders. supported by the National Special Support for R&D on Science and Technology for ITER (Ministry of Public Security of the People's Republic of China-MPS) (No. 2008GB102000)

Dry coal feeder development program at Ingersoll-Rand Research, Incorporated. [for coal gasification systems

NASA Technical Reports Server (NTRS)

Mistry, D. K.; Chen, T. N.

1977-01-01

A dry coal screw feeder for feeding coal into coal gasification reactors operating at pressures up to 1500 psig is described. Results on the feeder under several different modes of operation are presented. In addition, three piston feeder concepts and their technical and economical merits are discussed.

Using supplemental food and its influence on survival of northern bobwhite (Colinus virginianus)

USGS Publications Warehouse

Townsend, D.E.; Lochmiller, R.L.; DeMaso, S.J.; Leslie, David M.; Peoples, A.D.; Scott, A C.; Parry, E.S.

2000-01-01

Biologists have debated the effectiveness of supplemental feeders as a management tool for the northern bobwhite (Colinus virginianus), but few extensive evaluations have been conducted. We examined 783 crops from harvested bobwhites during 1992-1996 to determine effects of climatic stress in winter on use of supplemental feeders and their impact on survival rate in winter. Crops of bobwhites harvested from areas with supplemental feeders contained 28.2% supplemental food compared with 5.5% (P<0.001) for those from areas without supplemental feeders. Winter climate was not a significant predictor of the proportional use of supplemental feeders. Rates of winter survival were greater on areas with supplemental feeders compared with non-supplemented areas in winters 1992-1993 (P=0.001) and 1993-1994 (P=0.002), but in 1994-1995, rates were greater on nonsupplemented areas (P=0.032). Cause-specific mortality rates indicated that supplemental feeders did not predispose bobwhites to hunter harvest or predators. Results suggested that bobwhites can gain nutritional benefits from supplemental feeders during times of severe winter stress.

Exclusion of broiler breeder females from male feeders with a male only grill. 1. Behavioral and technical aspects of design and development.

PubMed

Brake, J; Khamidullin, T N; Fisinin, V I

1993-03-01

A series of experiments was conducted in order to develop a feeder grill that would allow broiler breeder males, but not females, to eat. Females are typically excluded from male feeders by increasing the height of the feeder. However, male feeder heights that exclude most females increase the time required for males to consume a given amount of feed 19 to 60%. Typical male feeder height from floor to feeder pan lip in commercial practice is 55 cm, which is about the distance from floor to the head of the male in an erect posture. Females measure about 40 cm in an erect posture. Because the necks of males (8.0 cm) are longer than those of females (6.3 cm), the lip of male feeder pans was extended horizontally 12.7 cm with a wire mesh on the assumption that females could not stretch their necks enough to reach the feed. However, the females learned quickly to perch on the extended lip and eat. Total exclusion of females from the male feeder, regardless of feeder height, was achieved by placing a horizontal upper mesh 5.6 to 10.2 cm above the extended lip, and connecting the upper mesh to the lower extended lip of the feeder with vertical bars spaced 5.1 cm apart. The horizontal upper mesh prevented perching by females and the 5.1-cm spacing of the vertical bars allowed males but not females to reach the feed by inserting their heads and necks up to their shoulders.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

PubMed Central

Wang, Xiaoyan; Chen, Tingfeng; Zhang, Yani; Li, Bichun; Xu, Qi; Song, Chengyi

2015-01-01

Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX) to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19). Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes. PMID:26556335

Periodic harvesting of embryonic stem cells from a hollow-fiber membrane based four-compartment bioreactor.

PubMed

Knöspel, Fanny; Freyer, Nora; Stecklum, Maria; Gerlach, Jörg C; Zeilinger, Katrin

2016-01-01

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers.

Defining the Regulation of Telomerase Through Identification of Mammary-Specific Telomerase Interacting Proteins

DTIC Science & Technology

2007-06-01

Pharmacology and Toxicology , Virginia Commonwealth University, Richmond, VA 2002-present Member, Molecular Biology and Genetics Program, Virginia...Studying Telomeres and Telomerase. Zebrafish , 1:349-355. Jones,K.R., L.W.Elmore, L.Povirk, S.E.Holt, and D.A.Gewirtz. 2005. Reciprocal regulation... Zebrafish Blastula Cell Line on Rainbow Trout Stromal Cells and Subsequent Development under Feeder-Free Conditions into a Cell Line, ZEB2J. Zebrafish 5: 49

Feeders of Free-Roaming Cats: Personal Characteristics, Feeding Practices, and Data on Cat Health and Welfare in an Urban Setting of Israel.

PubMed

Gunther, Idit; Raz, Tal; Even Zor, Yehonatan; Bachowski, Yuval; Klement, Eyal

2016-01-01

Cat feeders serve as an important source of available food for free-roaming cats (FRCs) and can play a central role in providing data on FRC distribution, welfare, and health. Data on cat feeder personalities as well as a better understanding of their feeding practices offer relevance for decision making concerning FRC population control strategies. The current study surveyed 222 FRC feeders who responded to a municipal trap-neuter-return (TNR) campaign in an Israeli central urban setting. The aim of the study was to describe their personal characteristics, feeding practices, and the FRC populations they feed. Feeders were divided into four groups according to the number of cats they claimed to feed per day (group 1: fed up to 5 cats, group 2: fed 6-10 cats, group 3: fed 11-20 cats, and group 4: fed ≥21 cats). Most feeders were women (81%), with a median age of 58 years (range 18-81). The feeders reported an overall feeding of 3337 cats in 342 different feeding locations. Feeders of group 4 comprised 15.31% (n = 34) of all feeders but fed 56% (n = 1869) of the FRC in 37.42% (n = 128) of the feeding locations. "Heavy" feeders (groups 3 and 4) reported that they traveled significantly longer distances in order to feed the cats. Commercial dry food consisted of 90% of the food they provided, with 66% of them feeding once a day, with less food per cat per day than the other feeder groups. Interestingly, "heavy" feeders were usually singles, had on average fewer siblings, a clear preference for owning cats as pets, and lived in lower income neighborhoods. According to the feeders' reports on the FRC populations they fed, 69.7% (2325/3337) cats were neutered and 11.8% (395/3337) were kittens. In addition, they reported that 1.6% (54/3337) of the cats were limping, 2% (67/3337) suffered from a systemic disease, 4% (135/3337) had skin lesions, and 3.9% (130/3337) were suffering from a chronic disability. Abundance of kittens and morbidity rate were significantly and negatively associated with neutering rate. These findings are in accordance with the suggestion that neutering may potentially improve cat welfare by reducing morbidity. Collaboration by the authorities with these heavy feeders, who represent a small number of FRC feeders and feed substantial FRC numbers, may be significant for the control and monitoring of FRC populations and their resources.

Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation.

PubMed

Ramírez, M Á; Pericuesta, E; Yáñez-Mó, M; Palasz, A; Gutiérrez-Adán, A

2011-02-01

Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. After 3 days culture or after long-term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT-mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long-term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long-term culture of mES cells in vitro. © 2010 Blackwell Publishing Ltd.

[Pooled Umbilical Cord Blood Plasma for Culturing UCMSC and Ex Vivo Expanding Umbilical Cord Blood CD34⁺ Cells].

PubMed

Wu, Jie-Ying; Lu, Yan; Chen, Jin-Song; Wu, Shao-Qing; Tang, Xue-Wei; Li, Yan

2015-08-01

To investigate the feasibility of umbilical cord blood plasma (UCP) as a replacement for fetal bovine serum (FBS) for culturing mesenchymal stem cells (MSC) derived from umbilical cord, and to observe the supporting effects of these cells (served as a feeder layer) on ex vivo expanding of human umbilical cord blood CD34(+) cells. Umbilical cord blood (UCB) units were suitable if the Guangzhou cord blood bank donor selection criteria strictly were fulfilled. UCP were ready to use after the collection from the plasma depletion/reduction during the processing and pooling of suitable UCB units (at least 30 units were screened for pathogens and microorganisms, and qualified). Umbilical cord mesenchymal stem cells (UCMSC) were harvested from the umbilical cord tissue of health full-term newborns after delivery by enzyme digestion and divided into 3 groups: group 1 and 2 were cultured in the presence of DMEM/F12 containing either FBS or UCP; and group 3 was cultured in serum-free medium (StemPro® MSC SFM CTS™). Morphology, proliferation and surface marker expression were examined by flow cytometry, and the differentiation toward adipogenic and osteogenic lineages was used for investigating the effect of media on UCMSC after 3-5 passages. Next, the cells cultured in the three different media were cryopreserved and thawed, then prepared as feeder layers with the name of UCMSC(FBS), UCMSC(UCP), and UCMSC(SFM), respectively. The CD34⁺ cells were separated from UCB by magnetic activated cell sorting (MACS) and divided into 4 groups cultured in StemPro(-34) SFM medium added with hematopoietic cytokine combination (StemSpan® CC100). The control group included only CD34⁺ cells as group A (blank control) and experimental groups included UCMSC(FBS) + CD34⁺ cells as group B, UCMSC(UCP) + CD34⁺ cells as group C, UCMSC(SFM) + CD34⁺ cells as group D, and cells in all groups were cultured ex vivo for 7 days. The nucleated cell (NC) number was counted by cell counter, CD34⁺ cells were measured by flow cytometry, and clonogenic assay was conducted at day 0 and 7 of culture. The expansion efficiency was assessed. The morphology (spindle-shaped and plastic-adherent), the immunophenotype (high positive percentage of CD73, CD90, CD105 and CD166) and the differentiation potential (osteogenic and adipogenic) were almost indistinguishable among the cells cultured in any of these three media except for the expression of CD105 in group 3 (serum-free medium) was lower than that in other 2 groups (P < 0.05). UCMSC grown in UCP medium demonstrated significantly higher proliferation rates than that in media containing FBS or commercial serum-free supplement (P < 0.05). After co-culture for 7 days, the CD34⁺ cell percentage decreased in all the groups, while NC were amplified effectively and the CD34⁺ cell number increased with the same order as group C or D group B or A (control group) (P < 0.05). As compared with the colony-forming unit (CFU) number at day 0, there was no significant difference in the expansion multiple between group C and D, while the expansion of CFU in group C were higher than that in group B and A. The UCP can be used as a better animal-free serum supplement for growth, maintenance and differentiation of UCMSC, thus would be a safe choice for clinical-scale production of human MSC.

30 CFR 75.1003-1 - Other requirements for guarding of trolley wires and trolley feeder wires.

Code of Federal Regulations, 2010 CFR

2010-07-01

... wires and trolley feeder wires. 75.1003-1 Section 75.1003-1 Mineral Resources MINE SAFETY AND HEALTH... Trolley Wires and Trolley Feeder Wires § 75.1003-1 Other requirements for guarding of trolley wires and trolley feeder wires. Adequate precaution shall be taken to insure that equipment being moved along...

30 CFR 75.1003-1 - Other requirements for guarding of trolley wires and trolley feeder wires.

Code of Federal Regulations, 2011 CFR

2011-07-01

... wires and trolley feeder wires. 75.1003-1 Section 75.1003-1 Mineral Resources MINE SAFETY AND HEALTH... Trolley Wires and Trolley Feeder Wires § 75.1003-1 Other requirements for guarding of trolley wires and trolley feeder wires. Adequate precaution shall be taken to insure that equipment being moved along...

Nanog is an essential factor for induction of pluripotency in somatic cells from endangered felids.

PubMed

Verma, Rajneesh; Liu, Jun; Holland, Michael Kenneth; Temple-Smith, Peter; Williamson, Mark; Verma, Paul John

2013-02-01

Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies.

Nanog Is an Essential Factor for Induction of Pluripotency in Somatic Cells from Endangered Felids

PubMed Central

Verma, Rajneesh; Liu, Jun; Holland, Michael Kenneth; Temple-Smith, Peter; Williamson, Mark

2013-01-01

Abstract Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies. PMID:23514873

Expansion and Differentiation of Germline-Derived Pluripotent Stem Cells on Biomaterials

PubMed Central

Šarić, Tomo; Denecke, Bernd; Peinkofer, Gabriel; Bovi, Manfred; Groll, Jürgen; Ko, Kinarm; Salber, Jochen; Halbach, Marcel; Schöler, Hans R.; Zenke, Martin; Neuss, Sabine

2013-01-01

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer® LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level. PMID:23234562

Expansion and differentiation of germline-derived pluripotent stem cells on biomaterials.

PubMed

Hoss, Mareike; Šarić, Tomo; Denecke, Bernd; Peinkofer, Gabriel; Bovi, Manfred; Groll, Jürgen; Ko, Kinarm; Salber, Jochen; Halbach, Marcel; Schöler, Hans R; Zenke, Martin; Neuss, Sabine

2013-05-01

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer(®) LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level.

Higher trophic level affects nutrient, silicon, metal(loid), and radionuclide mobilization from freshwater sediments

NASA Astrophysics Data System (ADS)

Schaller, Jörg; Planer-Friedrich, Britta

2017-04-01

Organic sediments in aquatic ecosystems are well known sinks for nutrients, silicon, and metal(loid)s. Organic matter-decomposing organisms like invertebrate shredders, grazers, bioturbators, and filter feeder are key-species for the carbon and energy turnover within the decomposer community. We could show that invertebrate shredders and grazer affect element fixation or remobilization by changing binding properties of organic sediments and the attached biofilm. Bioturbators affect element fixation or remobilization by changing redox conditions within the uppermost sediment layer. Last but not least filter feeders, like the zebra mussel Dreissena polymorpha, an invasive organism in North American and European freshwater ecosystems significantly contributed to element mobilization of silicon, iron, phosphorus, arsenic, and copper and to immobilization of uranium (p<0.001), probably driven by redox conditions, microbial activity within the gut system, or active control of element homeostasis. Except of the filter feeder D. polymorpha, the invertebrates are able to minimize the accumulation of non-nutrient elements due to specific strategies, which is an important strategy for species living in systems tending to element accumulation. However, D. polymorpha revealed a significant uptake and accumulation of arsenic, copper, iron, and especially uranium both into the soft body tissues and the seashell. This accumulation by D. polymorpha is in line with previous observations of metal(loid) accumulation from biomonitoring studies. In summary, higher trophic level strongly contributes to element fixation or remobilization in aquatic systems.

[Effects of canopy density on the functional group of soil macro fauna in Pinus massoniana plantations].

PubMed

Zhou, Hong Yang; Zhang, Dan Ju; Zhang, Jie; Zhao, Yan Bo; Zhao, Bo; Wei, Da Ping; Zhang, Jian

2017-06-18

In order to understand the effects of canopy density on the functional group characteristics of soil macrofauna in Pinus massoniana plantations, we divided the captured soil fauna into five types including xylophages, predators, saprophages, omnivores and fungal feeders. The results showed that 1) Saprozoic feeders had the highest percentage of total individuals, and the omnivores and xylophages occupied higher percentages of total taxa. 2) The individual and group number of the predators, and the group number of xylophages did not change significantly under 0.5-0.6 and then decreased significantly under 0.6-0.9 canopy density. 3) With the increasing canopy density, the individual an dgroup number of predators in litter layer decreased significantly, the saprozoic individual number in 5-10 cm soil layer represented irregular trends. The individual number of xylophage increased with the depth of soil, and the group number in litter layer, the individual and group number in 5-10 cm soil layer decreased significantly. 4) Pielou evenness of xylophage had no significant changes with the canopy density, all the other diversity index of xylophage and saprophage were various with the increasing canopy density. The predatory Simpson index was stable under 0.5-0.8, and then decreased significantly under 0.8-0.9 canopy density. 5) The CCA (canonical correlation analysis) indicated that soil bulk density and moisture content were the main environmental factors affecting functional groups of soil macro fauna. Moisture content greatly impacted on the number of saprophagous individuals. But xylophage and predators were mostly affected by soil bulk density, and the predatory Simpson index was mainly affected by soil pH value and total phosphorus. Our research indicated that the structure of soil macro faunal functional group under 0.7 canopy density was comparatively stable, which would facilitate the maintenance of soil fertility and ecological function in Pinus massoniana plantation.

Isolation and characterization of the trophectoderm from the Arabian camel (Camelus dromedarius).

PubMed

Saadeldin, Islam M; Swelum, Ayman Abdel-Aziz; Elsafadi, Mona; Moumen, Abdullah F; Alzahrani, Faisal A; Mahmood, Amer; Alfayez, Musaad; Alowaimer, Abdullah N

2017-09-01

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells

PubMed Central

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya

2015-01-01

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13+CD133+ cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13+CD133+ cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation. PMID:25808356

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells.

PubMed

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya; Kamiya, Akihide

2015-07-15

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13(+)CD133(+) cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13(+)CD133(+) cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation.

30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires § 75.1003...

30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires § 75.1003...

[Effects of prefrontal ablations on the reaction of the active choice of feeder under different probability and value of the reinforcement on dog].

PubMed

Preobrazhenskaia, L A; Ioffe, M E; Mats, V N

2004-01-01

The role of the prefrontal cortex was investigated on the reaction of the active choice of the two feeders under changes value and probability reinforcement. The experiments were performed on 2 dogs with prefrontal ablation (g. proreus). Before the lesions the dogs were taught to receive food in two different feeders to conditioned stimuli with equally probable alimentary reinforcement. After ablation in the inter-trial intervals the dogs were running from the one feeder to another. In the answer to conditioned stimuli for many times the dogs choose the same feeder. The disturbance of the behavior after some times completely restored. In the experiments with competition of probability events and values of reinforcement the dogs chose the feeder with low-probability but better quality of reinforcement. In the experiments with equal value but different probability the intact dogs chose the feeder with higher probability. In our experiments the dogs with prefrontal lesions chose the each feeder equiprobably. Thus in condition of free behavior one of different functions of the prefrontal cortex is the reactions choose with more probability of reinforcement.

Emplacement of Zebín Hill, Jičín Volcanic Field, Bohemian Paradise, Czech Republic: Anisotropy of Magnetic Susceptibility, Ground Magnetometry, Electric Resistivity Tomography, and Paleomagnetic Data

NASA Astrophysics Data System (ADS)

Petronis, M. S.; Rapprich, , V.; Valenta, J.; Leman, J.; Brister, A. R.; van Wyk de Vries, B.

2014-12-01

A well-preserved set of mid-Miocene tuff-cones and their feeders outcrop in the Jičín Volcanic Field, Czech Republic. Zebín Hill is a tuff cone that has been quarried to reveal the volcanoes feeder system. This edifice offers the opportunity to understand how magma is transported through a monogenetic pyroclastic cone. Rock types include a coarse-grained basal phreatomagmatic layer and a stratified upper wall facies both of which are penetrated by feeder dikes. Anisotropy of magnetic susceptibility (AMS) and paleomagnetic data were collected at twenty-one sites from feeder dikes and the main conduit of the volcano. A high-resolution ground magnetometry survey, electric resistivity tomography and seismic tomography were also conducted. Magnetic susceptibility intensity indicates that the dominant magnetic mineral is a ferromagnetic phase with little contribution from paramagnetic minerals. AMS ellipsoids shapes are both oblate and prolate and inferred magma flow directions indicate magma flow away from the central vent area and subhorizontal flow towards and away from the axial conduit; both upward and downward magma flow is evident at some sites. Curie point estimates yield a spectrum of results indicating a mixture of high-Ti titanomagnetite, iron sulfide, and low-Ti titanomagnetite. Ground magnetometry data indicate that both normal and reverse polarity rocks are present at Zebín Hill. Paleomagnetic data confirm the ground magnetic data in that both normal and reverse polarity rocks are present. Most sites yield a single component magnetization that is well grouped at the site level and carried by pseudosingle domain titanomagnetite. The presence of both normal and reverse polarity magnetizations from the volcano indicate that significant time passed during the growth of this monogenic system. Complex system of branching dikes has been also observed from electric resistivity tomography. The simple external structure of monogenetic volcanoes hides a rather complex magmatic plumbing system that dynamically evolves during the life of the volcano. As we show, the well-exposed roots of Zebín Hill reveals that the growth of a volcano occurs not due to simple central axis feeder systems but rather through interplay of local structures, magmatic effects, and construct evolution during the life of the volcano

Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture.

PubMed

Shaikh, Ambreen; Anand, Sandhya; Kapoor, Sona; Ganguly, Ranita; Bhartiya, Deepa

2017-04-01

Very small embryonic-like stem cells (VSELs) have been reported in various adult tissues, express pluripotent and primordial germ cells (PGCs) specific markers, are mobilized under stress/disease conditions, give rise to tissue committed progenitors and thus help regenerate and maintain homeostasis. The aim of the present study was to evaluate in vitro differentiation potential of VSELs using a quantitative approach. VSELs were collected from mouse bone marrow after 4 days of 5-fluorouracil (5-FU, 150 mg/Kg) treatment, further enriched by size based filtration and cultured on a feeder support in the presence of specific differentiation media. Cultured VSELs were found to differentiate into all three embryonic germ cell lineages, germ and hematopoietic cells after 14 days in culture. This was confirmed by studying Nestin, PDX-1, NKX2.5, DAZL, CD45 and other markers expression by various approaches. Very small, CD45 negative cells collected and enriched from GFP positive 5-FU treated mice bone marrow transitioned into CD45 positive cells in vitro thus demonstrating that VSELs can give rise to hematopoietic stem cells (HSCs). We envision that VSELs may be responsible for plasticity and ability of bone marrow cells to give rise to non-hematopoietic tissue progenitors of all 3 germ layers. Moreover the ability of VSELs to differentiate into germ cells as well as all the three lineages provides further evidence to support their pluripotent state and confirms developmental link between bone marrow VSELs and PGCs. The property of quiescence, no risk of teratoma formation and autologus source, make pluripotent VSELs a potential candidate to facilitate endogenous regeneration compared to cell replacement strategy envisioned using embryonic and induced pluripotent stem cells.

Binary colloidal crystals (BCCs) as a feeder-free system to generate human induced pluripotent stem cells (hiPSCs)

PubMed Central

Wang, Peng-Yuan; Hung, Sandy Shen-Chi; Thissen, Helmut; Kingshott, Peter; Wong, Raymond Ching-Bong

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into any cell type and provide significant advances to cell therapy and regenerative medicine. However, the current protocol for hiPSC generation is relatively inefficient and often results in many partially reprogrammed colonies, which increases the cost and reduces the applicability of hiPSCs. Biophysical stimulation, in particular from tuning cell-surface interactions, can trigger specific cellular responses that could in turn promote the reprogramming process. In this study, human fibroblasts were reprogrammed into hiPSCs using a feeder-free system and episomal vectors using novel substrates based on binary colloidal crystals (BCCs). BCCs are made from two different spherical particle materials (Si and PMMA) ranging in size from nanometers to micrometers that self-assemble into hexagonal close-packed arrays. Our results show that the BCCs, particularly those made from a crystal of 2 μm Si and 0.11 μm PMMA particles (2SiPM) facilitate the reprogramming process and increase the proportion of fully reprogrammed hiPSC colonies, even without a vitronectin coating. Subsequent isolation of clonal hiPSC lines demonstrates that they express pluripotent markers (OCT4 and TRA-1-60). This proof-of-concept study demonstrates that cell reprogramming can be improved on substrates where surface properties are tailored to the application. PMID:27833126

Numerical Modeling of Hailstorms and Hailstone Growth. Part III: Simulation of an Alberta Hailstorm--Natural and Seeded Cases.

NASA Astrophysics Data System (ADS)

Farley, Richard D.

1987-07-01

This paper reports on simulations of a multicellular hailstorm case observed during the 1983 Alberta Hail Project. The field operations on that day concentrated on two successive feeder cells which were subjected to controlled seeding experiments. The fist of these cells received the placebo treatment and the second was seeded with dry ice. The principal tool of this study is a modified version of the two-dimensional, time dependent hail category model described in Part I of this series of papers. It is with this model that hail growth processes are investigated, including the simulated effects of cloud seeding techniques as practiced in Alberta.The model simulation of the natural case produces a very good replication of the observed storm, particularly the placebo feeder cell. This is evidenced, in particular, by the high degree of fidelity of the observed and modeled radar reflectivity in terms of magnitudes, structure, and evolution. The character of the hailfall at the surface and the scale of the storm are captured nicely by the model, although cloud-top heights are generally too high, particularly for the mature storm system.Seeding experiments similar to those conducted in the field have also been simulated. These involve seeding the feeder cell early in its active development phase with dry ice (CO2) or silver iodide (AgI) introduced near cloud top. The model simulations of these seeded cases capture some of the observed seeding signatures detected by radar and aircraft. In these model experiments, CO2 seeding produced a stronger response than AgI seeding relative to inhibiting hail formation. For both seeded cases, production of precipitating ice was initially enhanced by the seeding, but retarded slightly in the later stages, the net result being modest increases in surface rainfall, with hail reduced slightly. In general, the model simulations support several subhypotheses of the operational strategy of the Alberta Research Council regarding the earlier formation of ice, snow, and graupel due to seeding.

Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors.

PubMed

Almutawaa, Walaa; Rohani, Leili; Rancourt, Derrick E

2016-01-01

Human induced pluripotent stem cells (hiPSCs) hold great promise as a cell source for therapeutic applications and regenerative medicine. Traditionally, hiPSCs are expanded in two-dimensional static culture as colonies in the presence or absence of feeder cells. However, this expansion procedure is associated with lack of reproducibility and low cell yields. To fulfill the large cell number demand for clinical use, robust large-scale production of these cells under defined conditions is needed. Herein, we describe a scalable, low-cost protocol for expanding hiPSCs as aggregates in a lab-scale bioreactor.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

PubMed Central

Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

2013-01-01

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

Characterization of an EPG waveform library for Lygus spp. on cotton squares

USDA-ARS?s Scientific Manuscript database

Lygus hesperus and L. lineolaris (Hemiptera: Miridae) are economically important pests affecting production of cotton in the western and mid-southern USA, respectively. Lygus feeding damage varies with instar; young nymphs are cell-rupture feeders performing laceration and maceration of plant tissue...

Continuation Power Flow Analysis for PV Integration Studies at Distribution Feeders

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

Wang, Jiyu; Zhu, Xiangqi; Lubkeman, David L.

2017-10-30

This paper presents a method for conducting continuation power flow simulation on high-solar penetration distribution feeders. A load disaggregation method is developed to disaggregate the daily feeder load profiles collected in substations down to each load node, where the electricity consumption of residential houses and commercial buildings are modeled using actual data collected from single family houses and commercial buildings. This allows the modeling of power flow and voltage profile along a distribution feeder on a continuing fashion for a 24- hour period at minute-by-minute resolution. By separating the feeder into load zones based on the distance between the loadmore » node and the feeder head, we studied the impact of PV penetration on distribution grid operation in different seasons and under different weather conditions for different PV placements.« less

Thermal Imaging for Assessment of Electron-Beam Free Form Fabrication (EBF(sup 3)) Additive Manufacturing Welds

NASA Technical Reports Server (NTRS)

Zalameda, Joseph N.; Burke, Eric R.; Hafley, Robert A.; Taminger, Karen M.; Domack, Christopher S.; Brewer, Amy R.; Martin, Richard E.

2013-01-01

Additive manufacturing is a rapidly growing field where 3-dimensional parts can be produced layer by layer. NASA s electron beam free-form fabrication (EBF(sup 3)) technology is being evaluated to manufacture metallic parts in a space environment. The benefits of EBF(sup 3) technology are weight savings to support space missions, rapid prototyping in a zero gravity environment, and improved vehicle readiness. The EBF(sup 3) system is composed of 3 main components: electron beam gun, multi-axis position system, and metallic wire feeder. The electron beam is used to melt the wire and the multi-axis positioning system is used to build the part layer by layer. To insure a quality weld, a near infrared (NIR) camera is used to image the melt pool and solidification areas. This paper describes the calibration and application of a NIR camera for temperature measurement. In addition, image processing techniques are presented for weld assessment metrics.

Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.

PubMed

Wang, Nan-Kai; Tosi, Joaquin; Kasanuki, Jennifer Mie; Chou, Chai Lin; Kong, Jian; Parmalee, Nancy; Wert, Katherine J; Allikmets, Rando; Lai, Chi-Chun; Chien, Chung-Liang; Nagasaki, Takayuki; Lin, Chyuan-Sheng; Tsang, Stephen H

2010-04-27

To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice. Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1.

PubMed

Javed, M Shahid; Yaqoob, Naeem; Iwamuro, Masaya; Kobayashi, Naoya; Fujiwara, Toshiyoshi

2014-02-01

To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. An experimental study. Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver-specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds.

Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

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

Yin, Xiaohui; Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191; Li, Yang

Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintainedmore » a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.« less

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

PubMed

Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

2011-06-01

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

A Batch Feeder for Inhomogeneous Bulk Materials

NASA Astrophysics Data System (ADS)

Vislov, I. S.; Kladiev, S. N.; Slobodyan, S. M.; Bogdan, A. M.

2016-04-01

The work includes the mechanical analysis of mechanical feeders and batchers that find application in various technological processes and industrial fields. Feeders are usually classified according to their design features into two groups: conveyor-type feeders and non-conveyor feeders. Batchers are used to batch solid bulk materials. Less frequently, they are used for liquids. In terms of a batching method, they are divided into volumetric and weighting batchers. Weighting batchers do not provide for sufficient batching accuracy. Automatic weighting batchers include a mass controlling sensor and systems for automatic material feed and automatic mass discharge control. In terms of operating principle, batchers are divided into gravitational batchers and batchers with forced feed of material using conveyors and pumps. Improved consumption of raw materials, decreased loss of materials, ease of use in automatic control systems of industrial facilities allows increasing the quality of technological processes and improve labor conditions. The batch feeder suggested by the authors is a volumetric batcher that has no comparable counterparts among conveyor-type feeders and allows solving the problem of targeted feeding of bulk material batches increasing reliability and hermeticity of the device.

Functional and aesthetic approach to design of bird feeders

NASA Astrophysics Data System (ADS)

Kukhta, A.; Kukhta, M.

2015-10-01

Anthropogenic objects which load the urban environment negatively affects the human psyche. The alternative is attracting elements of the natural environment into urban environment, of which some of the most frequently identified are birds. Attracting birds in the city is possible by means of feeders and artificial nests, however, both must be harmonious. The aim of this study is to analyze the essential functions of the feeders, and their integration into the environmental design and development of the city. On this basis an original feeder which is convenient for use by birds and attracts people's attention is developed. In this paper we apply comparative analysis of different types of feeders encountered in Tomsk, bird watching, and evaluate usability of different types of feeders from the position of their convenience both for birds and human beings. Historical-cultural analysis for determining features of the architectural and environmental design of Tomsk is carried out, the method allows us to solve engineering problems. In this study the feeder convenient for bird use is designed which blends harmoniously with the architectural design of Tomsk.

Modeling the Formation of Transverse Weld during Billet-on-Billet Extrusion

PubMed Central

Mahmoodkhani, Yahya; Wells, Mary; Parson, Nick; Jowett, Chris; Poole, Warren

2014-01-01

A comprehensive mathematical model of the hot extrusion process for aluminum alloys has been developed and validated. The plasticity module was developed using a commercial finite element package, DEFORM-2D, a transient Lagrangian model which couples the thermal and deformation phenomena. Validation of the model against industrial data indicated that it gave excellent predictions of the pressure during extrusion. The finite element predictions of the velocity fields were post-processed to calculate the thickness of the surface cladding as one billet is fed in after another through the die (i.e., the transverse weld). The mathematical model was then used to assess the effect a change in feeder dimensions would have on the shape, thickness and extent of the transverse weld during extrusion. Experimental measurements for different combinations of billet materials show that the model is able to accurately predict the transverse weld shape as well as the clad surface layer to thicknesses of 50 μm. The transverse weld is significantly affected by the feeder geometry shape, but the effects of ram speed, billet material and temperature on the transverse weld dimensions are negligible. PMID:28788629

Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

PubMed Central

Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2012-01-01

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500–2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation. PMID:22802633

Matrix remodeling maintains ESC self-renewal by activating Stat3

PubMed Central

Przybyla, Laralynne M.; Theunissen, Thorold W.; Jaenisch, Rudolf; Voldman, Joel

2013-01-01

While a variety of natural and synthetic matrices have been used to influence embryonic stem cell (ESC) self-renewal or differentiation, and ESCs also deposit a rich matrix of their own, the mechanisms behind how extracellular matrix affects cell fate are largely unexplored. The ESC matrix is continuously remodeled by matrix metalloproteinases (MMPs), a process that we find is enhanced by the presence of mouse embryonic fibroblast feeders in a paracrine manner. Matrix remodeling by MMPs aids in the self-renewal of ESCs, as inhibition of MMPs inhibits the ability of ESCs to self-renew. We also find that addition of the interstitial collagenase MMP1 is sufficient to maintain long-term LIF-independent mESC self-renewal in a dose-dependent manner. This remarkable ability is due to the presence of endogenously produced self-renewal-inducing signals, including the LIF-family ligand CNTF, that are normally trapped within the ECM and become exposed upon MMP-induced matrix remodeling to signal through JAK and Stat3. These results uncover a new role for feeder cells in maintaining self-renewal and show that mESCs normally produce sufficient levels of autocrine-acting pro-self-renewal ligands. PMID:23404867

Shared Ride Taxi Feeder Service in Memphis, TN

DOT National Transportation Integrated Search

1988-03-01

From May 1983 through October 1984, the Memphis Area Transit Authority (MATA) conducted the Taxi Feeder Demonstration Project. It entailed the operation of fixed-route feeder services through three low-density neighborhoods and one industrial park, c...

Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...

Hydrodynamics of microbial filter feeding

PubMed Central

Asadzadeh, Seyed Saeed; Dölger, Julia; Walther, Jens H.; Andersen, Anders

2017-01-01

Microbial filter feeders are an important group of grazers, significant to the microbial loop, aquatic food webs, and biogeochemical cycling. Our understanding of microbial filter feeding is poor, and, importantly, it is unknown what force microbial filter feeders must generate to process adequate amounts of water. Also, the trade-off in the filter spacing remains unexplored, despite its simple formulation: A filter too coarse will allow suitably sized prey to pass unintercepted, whereas a filter too fine will cause strong flow resistance. We quantify the feeding flow of the filter-feeding choanoflagellate Diaphanoeca grandis using particle tracking, and demonstrate that the current understanding of microbial filter feeding is inconsistent with computational fluid dynamics (CFD) and analytical estimates. Both approaches underestimate observed filtration rates by more than an order of magnitude; the beating flagellum is simply unable to draw enough water through the fine filter. We find similar discrepancies for other choanoflagellate species, highlighting an apparent paradox. Our observations motivate us to suggest a radically different filtration mechanism that requires a flagellar vane (sheet), something notoriously difficult to visualize but sporadically observed in the related choanocytes (sponges). A CFD model with a flagellar vane correctly predicts the filtration rate of D. grandis, and using a simple model we can account for the filtration rates of other microbial filter feeders. We finally predict how optimum filter mesh size increases with cell size in microbial filter feeders, a prediction that accords very well with observations. We expect our results to be of significance for small-scale biophysics and trait-based ecological modeling. PMID:28808016

Hydrodynamics of microbial filter feeding.

PubMed

Nielsen, Lasse Tor; Asadzadeh, Seyed Saeed; Dölger, Julia; Walther, Jens H; Kiørboe, Thomas; Andersen, Anders

2017-08-29

Microbial filter feeders are an important group of grazers, significant to the microbial loop, aquatic food webs, and biogeochemical cycling. Our understanding of microbial filter feeding is poor, and, importantly, it is unknown what force microbial filter feeders must generate to process adequate amounts of water. Also, the trade-off in the filter spacing remains unexplored, despite its simple formulation: A filter too coarse will allow suitably sized prey to pass unintercepted, whereas a filter too fine will cause strong flow resistance. We quantify the feeding flow of the filter-feeding choanoflagellate Diaphanoeca grandis using particle tracking, and demonstrate that the current understanding of microbial filter feeding is inconsistent with computational fluid dynamics (CFD) and analytical estimates. Both approaches underestimate observed filtration rates by more than an order of magnitude; the beating flagellum is simply unable to draw enough water through the fine filter. We find similar discrepancies for other choanoflagellate species, highlighting an apparent paradox. Our observations motivate us to suggest a radically different filtration mechanism that requires a flagellar vane (sheet), something notoriously difficult to visualize but sporadically observed in the related choanocytes (sponges). A CFD model with a flagellar vane correctly predicts the filtration rate of D. grandis , and using a simple model we can account for the filtration rates of other microbial filter feeders. We finally predict how optimum filter mesh size increases with cell size in microbial filter feeders, a prediction that accords very well with observations. We expect our results to be of significance for small-scale biophysics and trait-based ecological modeling.

The effect of space allowance and cage size on laying hens housed in furnished cages, Part II: Behavior at the feeder.

PubMed

Widowski, T M; Caston, L J; Casey-Trott, T M; Hunniford, M E

2017-09-01

Standards for feeder (a.k.a. feed trough) space allowance (SA) are based primarily on studies in conventional cages where laying hens tend to eat simultaneously, limiting feeder space. Large furnished cages (FC) offer more total space and opportunities to perform a greater variety of behaviors, which may affect feeding behavior and feeder space requirements. Our objective was to determine the effects of floor/feeder SA on behavior at the feeder. LSL-Lite hens were housed in FC equipped with a nest, perches, and a scratch mat. Hens with SA of either 520 cm2 (Low; 8.9 cm feeder space/hen) or 748 cm2 (High; 12.8 cm feeder space/hen) per bird resulted in groups of 40 vs. 28 birds in small FC (SFC) and 80 vs. 55 in large FC (LFC). Chain feeders ran at 0500, 0800, 1100, 1400, and 1700 with lights on at 0500 and off at 1900 hours. Digital recordings of FC were scanned at chain feeder onset and every 15 min for one h after (5 scans × 5 feeding times × 2 d) to count the number of birds with their head in the feeder. All occurrences of aggressive pecks and displacements during 2 continuous 30-minute observations at 0800 h and 1700 h also were counted. Mixed model repeated analyses tested the effects of SA, cage size, and time on the percent of hens feeding, and the frequency of aggressive pecks and displacements. Surprisingly, the percent of birds feeding simultaneously was similar regardless of cage size (LFC: 23.0 ± 0.9%; SFC: 24.0 ± 1.0%; P = 0.44) or SA (Low: 23.8 ± 0.9%; High: 23.3 ± 1.0%; P = 0.62). More birds were observed feeding at 1700 h (35.3 ± 0.1%) than any at other time (P < 0.001). Feeder use differed by cage area (nest, middle, or scratch) over the d (P < 0.001). The frequency of aggressive pecks was low overall and not affected by SA or cage size. Frequency of displacements was also low but greater at Low SA (P = 0.001). There was little evidence of feeder competition at the Low SA in this study. © The Author 2017. Published by Oxford University Press on behalf of Poultry Science Association.

Corneal repair by human corneal keratocyte-reprogrammed iPSCs and amphiphatic carboxymethyl-hexanoyl chitosan hydrogel.

PubMed

Chien, Yueh; Liao, Yi-Wen; Liu, Dean-Mo; Lin, Heng-Liang; Chen, Shih-Jen; Chen, Hen-Li; Peng, Chi-Hsien; Liang, Chang-Min; Mou, Chung-Yuan; Chiou, Shih-Hwa

2012-11-01

Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but whether iPSCs can promote corneal reconstruction remains undetermined. In this study, we successfully reprogrammed human corneal keratocytes into iPSCs. To prevent feeder cell contamination, these iPSCs were cultured onto a serum- and feeder-free system in which they remained stable through 30 passages and showed ESC-like pluripotent property. To investigate the availability of iPSCs as bioengineered substitutes in corneal repair, we developed a thermo-gelling injectable amphiphatic carboxymethyl-hexanoyl chitosan (CHC) nanoscale hydrogel and found that such gel increased the viability and CD44+proportion of iPSCs, and maintained their stem-cell like gene expression, in the presence of culture media. Combined treatment of iPSC with CHC hydrogel (iPSC/CHC hydrogel) facilitated wound healing in surgical abrasion-injured corneas. In severe corneal damage induced by alkaline, iPSC/CHC hydrogel enhanced corneal reconstruction by downregulating oxidative stress and recruiting endogenous epithelial cells to restore corneal epithelial thickness. Therefore, we demonstrated that these human keratocyte-reprogrammed iPSCs, when combined with CHC hydrogel, can be used as a rapid delivery system to efficiently enhance corneal wound healing. In addition, iPSCs reprogrammed from corneal surgical residues may serve as an alternative cell source for personalized therapies for human corneal damage. Copyright © 2012 Elsevier Ltd. All rights reserved.

39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. ...

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

39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. THE STAMP AN MORTAR BOX ARE ON THE LEFT AND THE FEEDER WITH ITS FEEDER DISK IS ON THE RIGHT. NOTE THE COLLAR ON THE CENTER STAMP STEM (UPPER LEFT CORNER OF THE IMAGE) THAT ACTIVATES THE LEVER IN THE CENTER OF THE PHOTO. THE COLLAR IS POSITIONED SUCH THAT WHEN THE LEVEL OF THE MATERIAL REACHES A LOW POINT IN THE MORTAR BOX IT PUSHES DOWN ON THE LEVER WHICH IN TURN ACTIVATES THE AUTOMATIC FEEDER DRIVE MECHANISM WHICH THEM DELIVERS ORE INTO THE BACKSIDE OF THE MORTAR BOX. - Standard Gold Mill, East of Bodie Creek, Northeast of Bodie, Bodie, Mono County, CA

A pellet feeder for the birds

PubMed Central

Millard, W. J.

1979-01-01

An alternative means of delivering food to pigeons in operant conditioning research is described. The feeder allows greater control of the amount of food delivered and reduces the amount of time necessary for the pigeon to collect the food. It is possible to extend the length of experimental sessions due to the reduction of food intake. Data obtained using the pellet feeder indicated that the control of responding is comparable to that observed with the standard grain-magazine feeder. PMID:16812118

Microscopic suspension feeders near boundaries: Effects of external water flow

NASA Astrophysics Data System (ADS)

Pepper, Rachel; Koehl, M. A. R.

2015-11-01

Microscopic sessile suspension feeders are an important part of aquatic ecosystems and form a vital link in the transfer of carbon in aquatic food webs. These suspension feeders live attached to boundaries, consume bacteria and small detritus, and are in turn eaten by larger organisms. Many create a feeding current that draws fluid towards them, and from which they filter their food. In still water, the feeding current consists of recirculating eddies which form as a result of fluid forcing near a boundary. These recirculating eddies can be depleted of food and significantly decrease nutrient uptake; a variety of strategies have been proposed for how attached feeders increase their access to undepleted water. We investigate the interaction of the flow produced by a microscopic suspension feeder with external environmental flow, such as the current in a stream or ocean. We show through calculations that even very slow flow (on the order of microns per second) is sufficient to provide a constant supply of undepleted water to suspension feeders when the feeders are modeled with perfect nutrient capture efficiency and in the absence of diffusion. We also discuss which natural flow environments exceed the threshold to supply undepleted water and which do not, and we examine how characteristics of the suspension feeders themselves, such as stalk length and feeding disk size, influence feeding currents and their interactions with external flows.

Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro.

PubMed

Martina, M N; Ramirez Bajo, M J; Bañon-Maneus, E; Moya Rull, D; Hierro-Garcia, N; Revuelta, I; Campistol, J M; Rovira, J; Diekmann, F

2016-11-01

Antibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. Human B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand-transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. Tofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non-class-switched memory B cells observed in nontreated cultures. The high predominance of CD27 + CD24 + phenotype was not modified by any immunosuppressive treatment. Our results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation. Copyright © 2016. Published by Elsevier Inc.

Flow accelerated corrosion of carbon steel feeder pipes from pressurized heavy water reactors

NASA Astrophysics Data System (ADS)

Singh, J. L.; Kumar, Umesh; Kumawat, N.; Kumar, Sunil; Kain, Vivekanand; Anantharaman, S.; Sinha, A. K.

2012-10-01

Detailed investigation of a number of feeder pipes received from Rajasthan Atomic Power Station Unit 2 (RAPS#2) after en-masse feeder pipe replacement after 15.67 Effective Full Power Years (EFPYs) was carried out. Investigations included ultrasonic thickness measurement by ultrasonic testing, optical microscopy, scanning electron microscopy, chemical analysis and X-ray Diffraction (XRD). Results showed that maximum thickness reduction of the feeder had occurred downstream and close to the weld in 32 NB (1.25″/32.75 mm ID) elbows. Rate of Flow Accelerated Corrosion (FAC) was measured to be higher in the lower diameter feeder pipes due to high flow velocity and turbulence. Weld regions had thinned to a lower extent than the parent material due to higher chromium content in the weld. A weld protrusion has been shown to add to the thinning due to FAC and lead to faster thinning rate at localized regions. Surface morphology of inner surface of feeder had shown different size scallop pattern over the weld and parent material. Inter-granular cracks were also observed along the weld fusion line and in the parent material in 32 NB outlet feeder elbow.

7 CFR 1280.107 - Feeder.

Code of Federal Regulations, 2010 CFR

2010-01-01

... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.107 Feeder. Feeder means any person who acquires ownership of lambs and feeds such lambs in the U.S. until they reach slaughter...

Apparatus and method for optimal phase balancing using dynamic programming with spatial consideration

DOEpatents

Robertazzi, Thomas G.; Skiena, Steven; Wang, Kai

2017-08-08

Provided are an apparatus and method for load-balancing of a three-phase electric power distribution system having a multi-phase feeder, including obtaining topology information of the feeder identifying supply points for customer loads and feeder sections between the supply points, obtaining customer information that includes peak customer load at each of the points between each of the feeder sections, performing a phase balancing analysis, and recommending phase assignment at the customer load supply points.

Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture.

PubMed

Bakhshi, Tiki; Zabriskie, Ryan C; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A; Gregory, Stephanie A; Fung, Henry C; Christopherson, Kent W

2008-12-01

Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow-derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton's jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. Umbilical cord-derived MSCs (UC-MSCs) were cultured from the Wharton's jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. These data suggest the potential therapeutic application of Wharton's jelly-derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.

Relative Preference and Localized Food Affect Predator Space Use and Consumption of Incidental Prey

PubMed Central

Schartel, Tyler E.; Schauber, Eric M.

2016-01-01

Abundant, localized foods can concentrate predators and their foraging efforts, thus altering both the spatial distribution of predation risk and predator preferences for prey that are encountered incidentally. However, few investigations have quantified the spatial scale over which localized foods affect predator foraging behavior and consumption of incidental prey. In spring 2010, we experimentally tested how point-source foods altered how generalist predators (white-footed mice, Peromyscus leucopus) utilized space and depredated two incidental prey items: almonds (Prunus dulcis; highly profitable) and maple seeds (Acer saccharum; less profitable). We estimated mouse population densities with trapping webs, quantified mouse consumption rates of these incidental prey items, and measured local mouse activity with track plates. We predicted that 1) mouse activity would be elevated near full feeders, but depressed at intermediate distances from the feeder, 2) consumption of both incidental prey would be high near feeders providing less-preferred food and, 3) consumption of incidental prey would be contingent on predator preference for prey relative to feeders providing more-preferred food. Mouse densities increased significantly from pre- to post-experiment. Mean mouse activity was unexpectedly greatest in control treatments, particularly <15 m from the control (empty) feeder. Feeders with highly preferred food (sunflower seeds) created localized refuges for incidental prey at intermediate distances (15 to 25m) from the feeder. Feeders with less-preferred food (corn) generated localized high risk for highly preferred almonds <10 m of the feeder. Our findings highlight the contingent but predictable effects of locally abundant food on risk experienced by incidental prey, which can be positive or negative depending on both spatial proximity and relative preference. PMID:26978659

Aflatoxin contamination in corn sold for wildlife feed in texas.

PubMed

Dunham, Nicholas R; Peper, Steven T; Downing, Carson D; Kendall, Ronald J

2017-05-01

Supplemental feeding with corn to attract and manage deer is a common practice throughout Texas. Other species, including northern bobwhites (Colinus virginianus), are commonly seen feeding around supplemental deer feeders. In many cases, supplemental feeding continues year-round so feed supply stores always have supplemental corn in stock. Fluctuating weather and improper storage of corn can lead to and/or amplify aflatoxin contamination. Due to the recent decline of bobwhites throughout the Rolling Plains ecoregion of Texas, there has been interest in finding factors such as toxins that could be linked to their decline. In this study, we purchased and sampled supplemental corn from 19 locations throughout this ecoregion to determine if aflatoxin contamination was present in individual bags prior to being dispersed to wildlife. Of the 57 bags sampled, 33 bags (approximately 58%) contained aflatoxin with a bag range between 0.0-19.91 parts per billion (ppb). Additionally, three metal and three polypropylene supplemental feeders were each filled with 45.4 kg of triple cleaned corn and placed in an open field to study long-term aflatoxin buildup. Feeders were sampled every 3 months from November 2013-November 2014. Average concentration of aflatoxin over the year was 4.08 ± 2.53 ppb (±SE) in metal feeders, and 1.43 ± 0.89 ppb (±SE) in polypropylene feeders. The concentration of aflatoxins is not affected by the type of feeder (metal vs polypropylene), the season corn was sampled, and the location in the feeder (top, middle, bottom) where corn is sampled. It is unlikely that corn used in supplemental feeders is contributing to the bobwhite decline due to the low levels of aflatoxin found in purchased corn and long-term storage of corn used in supplemental feeders.

Relative Preference and Localized Food Affect Predator Space Use and Consumption of Incidental Prey.

PubMed

Schartel, Tyler E; Schauber, Eric M

2016-01-01

Abundant, localized foods can concentrate predators and their foraging efforts, thus altering both the spatial distribution of predation risk and predator preferences for prey that are encountered incidentally. However, few investigations have quantified the spatial scale over which localized foods affect predator foraging behavior and consumption of incidental prey. In spring 2010, we experimentally tested how point-source foods altered how generalist predators (white-footed mice, Peromyscus leucopus) utilized space and depredated two incidental prey items: almonds (Prunus dulcis; highly profitable) and maple seeds (Acer saccharum; less profitable). We estimated mouse population densities with trapping webs, quantified mouse consumption rates of these incidental prey items, and measured local mouse activity with track plates. We predicted that 1) mouse activity would be elevated near full feeders, but depressed at intermediate distances from the feeder, 2) consumption of both incidental prey would be high near feeders providing less-preferred food and, 3) consumption of incidental prey would be contingent on predator preference for prey relative to feeders providing more-preferred food. Mouse densities increased significantly from pre- to post-experiment. Mean mouse activity was unexpectedly greatest in control treatments, particularly <15 m from the control (empty) feeder. Feeders with highly preferred food (sunflower seeds) created localized refuges for incidental prey at intermediate distances (15 to 25m) from the feeder. Feeders with less-preferred food (corn) generated localized high risk for highly preferred almonds <10 m of the feeder. Our findings highlight the contingent but predictable effects of locally abundant food on risk experienced by incidental prey, which can be positive or negative depending on both spatial proximity and relative preference.

Laboratory procedure for estimating residue dynamics of xenobiotic contaminants in a freshwater food chain

USGS Publications Warehouse

Johnson, B. Thomas

1980-01-01

A laboratory method of measuring the accumulation, transfer, elimination, and degradation of xenobiotic contaminants is described for organisms in a freshwater food chain (microorganisms, filter-feeder, and fish). A flow-through diluter-system, 14C-labeled contaminants, gas and thin-layer chromatography, autoradiography, and liquid scintillation spectrometry are used in making residue determinations. Accumulation factors and various index values are developed for measuring and estimating potential accumulation of xenobiotic contaminants by aquatic organisms. The laboratory procedure is economical, simple, reproducible, and ecologically relevant.

Voltage Support Study of Smart PV Inverters on a High-Photovoltaic Penetration Utility Distribution Feeder

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

Ding, Fei; Pratt, Annabelle; Bialek, Tom

2016-11-21

This paper reports on tools and methodologies developed to study the impact of adding rooftop photovoltaic (PV) systems, with and without the ability to provide voltage support, on the voltage profile of distribution feeders. Simulation results are provided from a study of a specific utility feeder. The simulation model of the utility distribution feeder was built in OpenDSS and verified by comparing the simulated voltages to field measurements. First, we set all PV systems to operate at unity power factor and analyzed the impact on feeder voltages. Then we conducted multiple simulations with voltage support activated for all the smartmore » PV inverters. These included different constant power factor settings and volt/VAR controls.« less

Analysis of broadcasting satellite service feeder link power control and polarization

NASA Technical Reports Server (NTRS)

Sullivan, T. M.

1982-01-01

Statistical analyses of carrier to interference power ratios (C/Is) were performed in assessing 17.5 GHz feeder links using (1) fixed power and power control, and (2) orthogonal linear and orthogonal circular polarizations. The analysis methods and attenuation/depolarization data base were based on CCIR findings to the greatest possible extent. Feeder links using adaptive power control were found to neither cause or suffer significant C/I degradation relative to that for fixed power feeder links having similar or less stringent availability objectives. The C/Is for sharing between orthogonal linearly polarized feeder links were found to be significantly higher than those for circular polarization only in links to nominally colocated satellites from nominally colocated Earth stations in high attenuation environments.

Network Reduction Algorithm for Developing Distribution Feeders for Real-Time Simulators

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

Nagarajan, Adarsh; Nelson, Austin A; Prabakar, Kumaraguru

As advanced grid-support functions (AGF) become more widely used in grid-connected photovoltaic (PV) inverters, utilities are increasingly interested in their impacts when implemented in the field. These effects can be understood by modeling feeders in real-time simulators and test PV inverters using power hardware-in-the-loop (PHIL) techniques. This paper presents a novel feeder model reduction algorithm using a ruin & reconstruct methodology that enables large feeders to be solved and operated on real-time computing platforms. Two Hawaiian Electric feeder models in Synergi Electric's load flow software were converted to reduced order models in OpenDSS, and subsequently implemented in the OPAL-RT real-timemore » digital testing platform. Smart PV inverters were added to the realtime model with AGF responses modeled after characterizing commercially available hardware inverters. Finally, hardware inverters were tested in conjunction with the real-time model using PHIL techniques so that the effects of AGFs on the feeders could be analyzed.« less

Network Reduction Algorithm for Developing Distribution Feeders for Real-Time Simulators: Preprint

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

Nagarajan, Adarsh; Nelson, Austin; Prabakar, Kumaraguru

As advanced grid-support functions (AGF) become more widely used in grid-connected photovoltaic (PV) inverters, utilities are increasingly interested in their impacts when implemented in the field. These effects can be understood by modeling feeders in real-time systems and testing PV inverters using power hardware-in-the-loop (PHIL) techniques. This paper presents a novel feeder model reduction algorithm using a Monte Carlo method that enables large feeders to be solved and operated on real-time computing platforms. Two Hawaiian Electric feeder models in Synergi Electric's load flow software were converted to reduced order models in OpenDSS, and subsequently implemented in the OPAL-RT real-time digitalmore » testing platform. Smart PV inverters were added to the real-time model with AGF responses modeled after characterizing commercially available hardware inverters. Finally, hardware inverters were tested in conjunction with the real-time model using PHIL techniques so that the effects of AGFs on the choice feeders could be analyzed.« less

Economic feeder for recharging and ``topping off''

NASA Astrophysics Data System (ADS)

Fickett, Bryan; Mihalik, G.

2000-04-01

Increasing the size of the melt charge significantly increases yield and reduces costs. Siemens Solar Industries is optimizing a method to charge additional material after meltdown (top-off) using an external feeder system. A prototype feeder system was fabricated consisting of a hopper and feed delivery system. The low-cost feeder is designed for simple operation and maintenance. The system is capable of introducing up to 60 kg of granular silicon while under vacuum. An isolation valve permits refilling of the hopper while maintaining vacuum in the growth furnace. Using the feeder system in conjunction with Siemens Solar Industries' energy efficient hot zone dramatically reduces power and argon consumption. Throughput is also improved as faster pull speeds can be attained. The increased pull speeds have an even greater impact when the charge size is increased. Further cost reduction can be achieved by refilling the crucible after crystal growth and pulling a second ingot run. Siemens Solar Industries is presently testing the feeder in production.

Comparing the 2000 and 2005 factors affecting the selling price of feeder cattle sold at Arkansas livestock auctions.

PubMed

Troxel, T R; Barham, B L

2007-12-01

The objectives of the study were to determine how factors affecting the selling price of feeder calves changed from 2000 to 2005 and to examine the perception that discounts narrow or even disappear as calf supplies decrease and selling prices increase. Data from weekly Arkansas livestock auctions were collected from January 1 to December 31 in 2000 and 2005. Data included calf sex, breed type, color, muscle score, horn status, frame score, fill, condition, health, and BW. Mean selling prices for 2000 and 2005 were $92.91 +/- 15.05 and $118.32 +/- 15.13 (mean +/- SD; $/45.45 kg), respectively. Individual price observations were subtracted from the respective annual means and became the dependent variable. The selling prices for feeder calves sold in groups of 2 to 5 calves and in groups of >/= 6 calves were greater in 2005 than 2000 (P < 0.001). Steers received a greater premium ($6.48 +/- 0.09 vs. $6.02 +/- 0.08; mean +/- SE) and bull calves received greater discounts ($0.30 +/- 0.14 vs. $1.68 +/- 0.09) in 2005 than in 2000. Breeds types that increased in value from 2000 to 2005 were Angus x Hereford, Angus, Angus x Charolais, and Brahman (P < 0.001). Breed types that received a reduced selling price in 2005 compared with 2000 (P < 0.001) were one-fourth Brahman Cross, Charolais, Charolais x Limousin, Hereford x Limousin, Limousin, Limousin x one-fourth Brahman, Longhorn, Saler and Simmental. Yellow-white face, black-white face, black, and gray feeder calves received an increase in selling price from 2000 to 2005 (P < 0.001). Although fewer horned feeder calves were sold in 2005 (P < 0.01), they received greater discounts in 2005 than 2000 (-$2.86 +/- 0.16 and -$0.51 +/- 0.09; P < 0.001). In 2005, large-framed feeder calves did not receive the premium detected in 2000, but medium-framed feeder calves in 2005 received a greater selling price compared with 2000. Feeder calves with a muscle score of 1 received a greater premium in 2005 compared with 2000 ($2.58 +/- 0.06 and $0.02 +/- 0.09, respectively; P < 0.001). Feeder calves with a muscle score of 2 were discounted in both years, but the discount in 2005 was not as great as in 2000 (P < 0.001). Full and tanked feeder calves received greater discounts in 2005 than in 2000 (P < 0.001). Discounts for fleshy and fat feeder calves were greater in 2005 than in 2000. Most factors affecting the selling price of Arkansas feeder calves in 2000 affected the selling price in 2005. Although feeder calf supplies were smaller in 2005 than 2000, many discounts increased.

An RFID Based Smart Feeder for Hummingbirds.

PubMed

Ibarra, Vicente; Araya-Salas, Marcelo; Tang, Yu-ping; Park, Charlie; Hyde, Anthony; Wright, Timothy F; Tang, Wei

2015-12-16

We present an interdisciplinary effort to record feeding behaviors and control the diet of a hummingbird species (Phaethornis longirostris, the long-billed hermit or LBH) by developing a Radio Frequency Identification (RFID) based smart feeder. The system contains an RFID reader, a microcontroller, and a servo-controlled hummingbird feeder opener; the system is presented as a tool for studying the cognitive ability of the LBH species. When equipped with glass capsule RFID tags (which are mounted on the hummingbird), the smart feeder can provide specific diets for predetermined sets of hummingbirds at the discretion of biologists. This is done by reading the unique RFID tag on the hummingbirds and comparing the ID number with the pre-programmed ID numbers stored in the smart feeder. The smart feeder records the time and ID of each hummingbird visit. The system data is stored in a readily available SD card and is powered by two 9 V batteries. The detection range of the system is approximately 9-11 cm. Using this system, biologists can assign the wild hummingbirds to different experimental groups and monitor their diets to determine if they develop a preference to any of the available nectars. During field testing, the smart feeder system has demonstrated consistent detection (when compared to detections observed by video-recordings) of RFID tags on hummingbirds and provides pre-designed nectars varying water and sugar concentrations to target individuals. The smart feeder can be applied to other biological and environmental studies in the future.

An RFID Based Smart Feeder for Hummingbirds

PubMed Central

Ibarra, Vicente; Araya-Salas, Marcelo; Tang, Yu-ping; Park, Charlie; Hyde, Anthony; Wright, Timothy F.; Tang, Wei

2015-01-01

We present an interdisciplinary effort to record feeding behaviors and control the diet of a hummingbird species (Phaethornis longirostris, the long-billed hermit or LBH) by developing a Radio Frequency Identification (RFID) based smart feeder. The system contains an RFID reader, a microcontroller, and a servo-controlled hummingbird feeder opener; the system is presented as a tool for studying the cognitive ability of the LBH species. When equipped with glass capsule RFID tags (which are mounted on the hummingbird), the smart feeder can provide specific diets for predetermined sets of hummingbirds at the discretion of biologists. This is done by reading the unique RFID tag on the hummingbirds and comparing the ID number with the pre-programmed ID numbers stored in the smart feeder. The smart feeder records the time and ID of each hummingbird visit. The system data is stored in a readily available SD card and is powered by two 9 V batteries. The detection range of the system is approximately 9–11 cm. Using this system, biologists can assign the wild hummingbirds to different experimental groups and monitor their diets to determine if they develop a preference to any of the available nectars. During field testing, the smart feeder system has demonstrated consistent detection (when compared to detections observed by video-recordings) of RFID tags on hummingbirds and provides pre-designed nectars varying water and sugar concentrations to target individuals. The smart feeder can be applied to other biological and environmental studies in the future. PMID:26694402

Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

DOE PAGES

Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; ...

2015-12-22

Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

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

Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.

Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

46 CFR 111.75-1 - Lighting feeders.

Code of Federal Regulations, 2010 CFR

2010-10-01

... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting feeders. (a) Passenger vessels. On a... lighting, feeders, and branch circuits are in subpart 112.43 of this chapter. [CGD 74-125A, 47 FR 15236...

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells.

PubMed

Kumar, R; Ahlawat, S P S; Sharma, M; Verma, O P; Sai Kumar, G; Taru Sharma, G

2014-03-01

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

46 CFR 112.43-15 - Emergency lighting feeders.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 4 2011-10-01 2011-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with...

46 CFR 112.43-15 - Emergency lighting feeders.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with...

46 CFR 112.43-15 - Emergency lighting feeders.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with...

46 CFR 112.43-15 - Emergency lighting feeders.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with...

46 CFR 112.43-15 - Emergency lighting feeders.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with...

77 FR 34876 - Airworthiness Directives; The Boeing Company

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-12

... (a flammable fluid leakage zone) or heat damage to the APU power feeder cable, insulation blankets... heat damage to the APU power feeder cable, insulation blankets, or pressure bulkhead. Relevant Service... feeder cable and heat damage of the insulation blanket adjacent to the clamp, a detailed inspection for...

Feederism in a woman.

PubMed

Terry, Lesley L; Vasey, Paul L

2011-06-01

Feederism is a fat fetish subculture in which individuals eroticize weight gain and feeding. Feeders are individuals who claim to become sexually aroused by feeding their partners and encouraging them to gain weight. Conversely, Feedees are individuals who claim to become sexually aroused by eating, being fed, and the idea or act of gaining weight. Very little is known about this population. This report describes a woman who self-identified as a Feedee. It is unclear, at present, whether female Feederism represents a unique paraphilia or a thematic variation of morphophilia or sexual masochism.

Changes in food intake and abnormal behavior using a puzzle feeder in newly acquired sub-adult rhesus monkeys (Macaca mulatta): a short term study.

PubMed

Lee, Jae-Il; Lee, Chi-Woo; Kwon, Hyouk-Sang; Kim, Young-Tae; Park, Chung-Gyu; Kim, Sang-Joon; Kang, Byeong-Cheol

2008-10-01

The majority of newly acquired nonhuman primates encounter serious problems adapting themselves to new environments or facilities. In particular, loss of appetite and abnormal behavior can occur in response to environmental stresses. These adaptation abnormalities can ultimately have an affect on the animal's growth and well-being. In this study, we evaluated the affects of a puzzle feeder on the food intake and abnormal behavior of newly acquired rhesus monkeys for a short period. The puzzle feeder was applied to 47- to 58-month-old animals that had never previously encountered one. We found that there was no difference in the change of food intake between the bucket condition and the puzzle feeder condition. In contrast, the time spent for consumption of food was three times longer in the puzzle feeder condition than in the bucket condition. Two monkeys initially exhibited stereotypic behavior. One showed a decreasing, and the other an increasing pattern of abnormal behavior after introduction of the puzzle feeder. In conclusion, this result suggests that over a short period, the puzzle feeder can only affect the time for food consumption since it failed to affect the food intake and did not consistently influence stereotypic behaviors in newly acquired rhesus monkeys.

Differentiation of human pluripotent stem cells into highly functional classical brown adipocytes.

PubMed

Nishio, Miwako; Saeki, Kumiko

2014-01-01

We describe a detailed method for directed differentiation of human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), into functional classical brown adipocytes (BAs) under serum-free and feeder-free conditions. It is a two-tiered culture system, based on very simple techniques, a floating culture and a subsequent adherent culture. It does not require gene transfer. The entire process can be carried out in about 10 days. The key point is the usage of our special hematopoietic cytokine cocktail. Almost all the differentiated cells express uncoupling protein 1, a BA-selective marker, as determined by immunostaining. The differentiated cells show characteristics of classical BA as assessed by morphology and gene/protein expression. Moreover, the expression of myoblast marker genes is transiently induced during the floating culture step. hESC/hiPSC-derived BAs show significantly higher oxygen consumption rates (OCRs) than white adipocytes generated from human mesenchymal stem cell. They also show responsiveness to adrenergic stimuli, with about twofold upregulation in OCR by β-adrenergic receptor (β-AR) agonist treatments. hESC/hiPSC-derived BAs exert in vivo calorigenic activities in response to β-AR agonist treatments as assessed by thermography. Finally, lipid and glucose metabolisms are significantly improved in hESC/hiPSC-derived BA-transplanted mice. Our system provides a highly feasible way to produce functional classical BA bearing metabolism-improving capacities from hESC/hiPSC under a feeder-free and serum-free condition without gene transfer. © 2014 Elsevier Inc. All rights reserved.

Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

PubMed

Talbot, N C; Caperna, T J; Garrett, W M

2013-01-01

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.

The innate responses of bumble bees to flower patterns: separating the nectar guide from the nectary changes bee movements and search time

NASA Astrophysics Data System (ADS)

Goodale, Eben; Kim, Edward; Nabors, Annika; Henrichon, Sara; Nieh, James C.

2014-06-01

Nectar guides can enhance pollinator efficiency and plant fitness by allowing pollinators to more rapidly find and remember the location of floral nectar. We tested if a radiating nectar guide around a nectary would enhance the ability of naïve bumble bee foragers to find nectar. Most experiments that test nectar guide efficacy, specifically radiating linear guides, have used guides positioned around the center of a radially symmetric flower, where nectaries are often found. However, the flower center may be intrinsically attractive. We therefore used an off-center guide and nectary and compared "conjunct" feeders with a nectar guide surrounding the nectary to "disjunct" feeders with a nectar guide separated from the nectary. We focused on the innate response of novice bee foragers that had never previously visited such feeders. We hypothesized that a disjunct nectar guide would conflict with the visual information provided by the nectary and negatively affect foraging. Approximately, equal numbers of bumble bees ( Bombus impatiens) found nectar on both feeder types. On disjunct feeders, however, unsuccessful foragers spent significantly more time (on average 1.6-fold longer) searching for nectar than any other forager group. Successful foragers on disjunct feeders approached these feeders from random directions unlike successful foragers on conjunct feeders, which preferentially approached the combined nectary and nectar guide. Thus, the nectary and a surrounding nectar guide can be considered a combination of two signals that attract naïve foragers even when not in the floral center.

The innate responses of bumble bees to flower patterns: separating the nectar guide from the nectary changes bee movements and search time.

PubMed

Goodale, Eben; Kim, Edward; Nabors, Annika; Henrichon, Sara; Nieh, James C

2014-06-01

Nectar guides can enhance pollinator efficiency and plant fitness by allowing pollinators to more rapidly find and remember the location of floral nectar. We tested if a radiating nectar guide around a nectary would enhance the ability of naïve bumble bee foragers to find nectar. Most experiments that test nectar guide efficacy, specifically radiating linear guides, have used guides positioned around the center of a radially symmetric flower, where nectaries are often found. However, the flower center may be intrinsically attractive. We therefore used an off-center guide and nectary and compared "conjunct" feeders with a nectar guide surrounding the nectary to "disjunct" feeders with a nectar guide separated from the nectary. We focused on the innate response of novice bee foragers that had never previously visited such feeders. We hypothesized that a disjunct nectar guide would conflict with the visual information provided by the nectary and negatively affect foraging. Approximately, equal numbers of bumble bees (Bombus impatiens) found nectar on both feeder types. On disjunct feeders, however, unsuccessful foragers spent significantly more time (on average 1.6-fold longer) searching for nectar than any other forager group. Successful foragers on disjunct feeders approached these feeders from random directions unlike successful foragers on conjunct feeders, which preferentially approached the combined nectary and nectar guide. Thus, the nectary and a surrounding nectar guide can be considered a combination of two signals that attract naïve foragers even when not in the floral center.

26 CFR 1.502-1 - Feeder organizations.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Feeder organizations. 1.502-1 Section 1.502-1...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.502-1 Feeder organizations. (a) In the case of an organization operated for the primary purpose of carrying on a trade or business for profit, exemption is not...

75 FR 8395 - Bunker Hill Groundwater Basin, Riverside-Corona Feeder Project, San Bernardino and Riverside...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-24

... DEPARTMENT OF THE INTERIOR Bureau of Reclamation Bunker Hill Groundwater Basin, Riverside-Corona...) will prepare a joint EIS/EIR for the proposed Riverside-Corona Feeder Project. The public and agencies... participate in the planning, design, and construction of the Riverside-Corona Feeder Project including: (i) 20...

7 CFR 1230.71 - Assessments.

Code of Federal Regulations, 2011 CFR

2011-01-01

... feeder pig that is sold shall pay an assessment on that animal, unless such producer demonstrates to the Board by appropriate documentation that an assessment was previously paid on that animal as a feeder pig... purchaser of a porcine animal raised by a producer as a feeder pig or market hog shall collect an assessment...

7 CFR 1230.71 - Assessments.

Code of Federal Regulations, 2010 CFR

2010-01-01

... feeder pig that is sold shall pay an assessment on that animal, unless such producer demonstrates to the Board by appropriate documentation that an assessment was previously paid on that animal as a feeder pig... purchaser of a porcine animal raised by a producer as a feeder pig or market hog shall collect an assessment...

7 CFR 1230.71 - Assessments.

Code of Federal Regulations, 2012 CFR

2012-01-01

... feeder pig that is sold shall pay an assessment on that animal, unless such producer demonstrates to the Board by appropriate documentation that an assessment was previously paid on that animal as a feeder pig... purchaser of a porcine animal raised by a producer as a feeder pig or market hog shall collect an assessment...

7 CFR 1230.71 - Assessments.

Code of Federal Regulations, 2014 CFR

2014-01-01

... feeder pig that is sold shall pay an assessment on that animal, unless such producer demonstrates to the Board by appropriate documentation that an assessment was previously paid on that animal as a feeder pig... purchaser of a porcine animal raised by a producer as a feeder pig or market hog shall collect an assessment...

7 CFR 1230.71 - Assessments.

Code of Federal Regulations, 2013 CFR

2013-01-01

... feeder pig that is sold shall pay an assessment on that animal, unless such producer demonstrates to the Board by appropriate documentation that an assessment was previously paid on that animal as a feeder pig... purchaser of a porcine animal raised by a producer as a feeder pig or market hog shall collect an assessment...

A fundamental dispute: A discussion of "On some fundamentals of igneous petrology" by Bruce D. Marsh, Contributions to Mineralogy and Petrology (2013) 166: 665-690

NASA Astrophysics Data System (ADS)

Latypov, Rais; Morse, Tony; Robins, Brian; Wilson, Richard; Cawthorn, Grant; Tegner, Christian; Holness, Marian; Lesher, Charles; Barnes, Steve; O'Driscoll, Brian; Veksler, Ilya; Higgins, Michael; Wilson, Allan; Namur, Olivier; Chistyakova, Sofya; Naslund, Richard; Thy, Peter

2015-02-01

Marsh (Contrib Miner Petrol 166:665-690, 2013) again claims that crystal-free basalt magmas are unable to differentiate in crustal magma chambers and regards layered intrusions as primarily due to the repeated emplacement of crystal suspensions. He ignores an earlier critique of his unconventional inferences (Latypov, J Petrol 50:1047-1069, 2009) as well as a wealth of petrographic, geochemical and experimental evidence supporting the dominant role of fractional crystallization in the solidification of layered intrusions. Most tellingly, the cryptic variations preserved in the Skaergaard and many other basaltic layered intrusions would require an exceedingly implausible sequence of phenocrystic magmas but are wholly consistent with in situ fractional crystallization. A major flaw in Marsh's hypothesis is that it dismisses progressive fractional crystallization within any magma chamber and hence prohibits the formation of crystal slurries with phenocrysts and melts that change systematically in composition in any feeder system. This inherent attribute of the hypothesis excludes the formation of layered intrusions anywhere.

A Multistate Investigation of Antibiotic-Resistant Salmonella enterica Serotype I 4,[5],12:i:- Infections as Part of an International Outbreak Associated with Frozen Feeder Rodents

PubMed Central

Cartwright, E. J.; Nguyen, T.; Melluso, C.; Ayers, T.; Lane, C.; Hodges, A.; Li, X.; Quammen, J.; Yendell, S. J.; Adams, J.; Mitchell, J.; Rickert, R.; Klos, R.; Williams, I. T.; Behravesh, C. Barton; Wright, J.

2015-01-01

While most human Salmonella infections result from exposure to contaminated foods, an estimated 11% of all Salmonella infections are attributed to animal exposures, including both direct animal handling and indirect exposures such as cleaning cages and handling contaminated pet food. This report describes the epidemiologic, environmental and laboratory investigations conducted in the United States as part of the response to an international outbreak of tetracycline-resistant Salmonella enterica serotype I 4,[5],12:i:- infections with over 500 illnesses occurring from 2008 to 2010. This investigation found that illness due to the outbreak strain was significantly associated with exposure to pet reptiles and frozen feeder rodents used as food for pet reptiles. Salmonella isolates indistinguishable from the outbreak strain were isolated from a frozen feeder mice-fed reptile owned by a case patient, as well as from frozen feeder mice and environmental samples collected from a rodent producing facility (Company A). An international voluntary recall of all Company A produced frozen feeder animals sold between May 2009 and July 2010 occurred. Only 13% of cases in our investigation were aware of the association between Salmonella infection and mice or rats. Consumers, the pet industry, healthcare providers and veterinarians need to be aware of the potential health risk posed by feeder rodents, whether live or frozen. Frozen feeder rodent producers, suppliers and distributors should follow the animal food labelling requirements as described in 21 CFR §501.5, and all packages of frozen feeder rodents should include safe handling instructions. Persons should wash their hands thoroughly with soap and water after handling live or frozen feeder rodents, as well as reptiles or anything in the area where the animals live. Continued opportunities exist for public health officials, the pet industry, veterinarians and consumers to work together to prevent salmonellosis associated with pet food, pets and other animals. PMID:25996458

A Multistate Investigation of Antibiotic-Resistant Salmonella enterica Serotype I 4,[5],12:i:- Infections as Part of an International Outbreak Associated with Frozen Feeder Rodents.

PubMed

Cartwright, E J; Nguyen, T; Melluso, C; Ayers, T; Lane, C; Hodges, A; Li, X; Quammen, J; Yendell, S J; Adams, J; Mitchell, J; Rickert, R; Klos, R; Williams, I T; Barton Behravesh, C; Wright, J

2016-02-01

While most human Salmonella infections result from exposure to contaminated foods, an estimated 11% of all Salmonella infections are attributed to animal exposures, including both direct animal handling and indirect exposures such as cleaning cages and handling contaminated pet food. This report describes the epidemiologic, environmental and laboratory investigations conducted in the United States as part of the response to an international outbreak of tetracycline-resistant Salmonella enterica serotype I 4,[5],12:i:- infections with over 500 illnesses occurring from 2008 to 2010. This investigation found that illness due to the outbreak strain was significantly associated with exposure to pet reptiles and frozen feeder rodents used as food for pet reptiles. Salmonella isolates indistinguishable from the outbreak strain were isolated from a frozen feeder mice-fed reptile owned by a case patient, as well as from frozen feeder mice and environmental samples collected from a rodent producing facility (Company A). An international voluntary recall of all Company A produced frozen feeder animals sold between May 2009 and July 2010 occurred. Only 13% of cases in our investigation were aware of the association between Salmonella infection and mice or rats. Consumers, the pet industry, healthcare providers and veterinarians need to be aware of the potential health risk posed by feeder rodents, whether live or frozen. Frozen feeder rodent producers, suppliers and distributors should follow the animal food labelling requirements as described in 21 CFR §501.5, and all packages of frozen feeder rodents should include safe handling instructions. Persons should wash their hands thoroughly with soap and water after handling live or frozen feeder rodents, as well as reptiles or anything in the area where the animals live. Continued opportunities exist for public health officials, the pet industry, veterinarians and consumers to work together to prevent salmonellosis associated with pet food, pets and other animals. © 2015 Blackwell Verlag GmbH.

A metro-access integrated network with all-optical virtual private network function using DPSK/ASK modulation format

NASA Astrophysics Data System (ADS)

Tian, Yue; Leng, Lufeng; Su, Yikai

2008-11-01

All-optical virtual private network (VPN), which offers dedicated optical channels to connect users within a VPN group, is considered a promising approach to efficient internetworking with low latency and enhanced security implemented in the physical layer. On the other hand, time-division multiplexed (TDM) / wavelength-division multiplexed (WDM) network architecture based on a feeder-ring with access-tree topology, is considered a pragmatic migration scenario from current TDM-PONs to future WDM-PONs and a potential convergence scheme for access and metropolitan networks, due to its efficiently shared hardware and bandwidth resources. All-optical VPN internetworking in such a metro-access integrated structure is expected to cover a wider service area and therefore is highly desirable. In this paper, we present a TDM/WDM metro-access integrated network supporting all-optical VPN internetworking among ONUs in different sub- PONs based on orthogonal differential-phase-shift keying (DPSK) / amplitude-shift keying (ASK) modulation format. In each ONU, no laser but a single Mach-Zehnder modulator (MZM) is needed for the upstream and VPN signal generation, which is cost-effective. Experiments and simulations are performed to verify its feasibility as a potential solution to the future access service.

Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

PubMed

Dahlmann, Julia; Awad, George; Dolny, Carsten; Weinert, Sönke; Richter, Karin; Fischer, Klaus-Dieter; Munsch, Thomas; Leßmann, Volkmar; Volleth, Marianne; Zenker, Martin; Chen, Yaoyao; Merkl, Claudia; Schnieke, Angelika; Baraki, Hassina; Kutschka, Ingo; Kensah, George

2018-01-01

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

Development of dry coal feeders

NASA Technical Reports Server (NTRS)

Bonin, J. H.; Cantey, D. E.; Daniel, A. D., Jr.; Meyer, J. W.

1977-01-01

Design and fabrication of equipment of feed coal into pressurized environments were investigated. Concepts were selected based on feeder system performance and economic projections. These systems include: two approaches using rotating components, a gas or steam driven ejector, and a modified standpipe feeder concept. Results of development testing of critical components, design procedures, and performance prediction techniques are reviewed.

Faculty Articulation with Feeder High Schools and Local Employers.

ERIC Educational Resources Information Center

Parrott, Marietta

As a first step in developing an articulation plan with feeder high schools, a College of the Sequoias (COS) task force developed and distributed a survey to all full-time faculty members to determine if individual faculty members were articulating with feeder high schools and local businesses, and if they would be willing to participate in an…

Ornithologists by Design: Kindergarteners Design, Construct, and Evaluate Bird Feeders

ERIC Educational Resources Information Center

Shorter, Angela; Segers, Marcia

2016-01-01

How can an engineer design a bird feeder that attracts many birds? This question resulted from kindergarten students' observations of the bird feeders in their school's bird sanctuary. The challenging question is the heart of project-based learning (PBL), a teaching strategy in which students tackle real-world problems and design projects to solve…

Preliminary evaluation of feeder and lint slide moisture addition on ginning, fiber quality, and textile processing of western cotton

USDA-ARS?s Scientific Manuscript database

The objective of this study was to evaluate the effects of moisture addition at the gin stand feeder conditioning hopper and/or the battery condenser slide on gin performance and Western cotton fiber quality and textile processing. The test treatments included no moisture addition, feeder hopper hum...

19. DETAIL OF STAMP BATTERY AUTOMATIC FEEDER, LOOKING EAST. THIS ...

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

19. DETAIL OF STAMP BATTERY AUTOMATIC FEEDER, LOOKING EAST. THIS IS THE MIDDLE OF THREE FEEDERS, ONE FOR EACH STAMP BATTERY. THE CHUTE (UPPER RIGHT) INTRODUCED THE CRUSHED ORE FROM THE ORE BIN. FLOW WAS CONTROLLED BY A SLIDING DOOR ON THE UPPER LEVEL. - Skidoo Mine, Park Route 38 (Skidoo Road), Death Valley Junction, Inyo County, CA

Impact of feeder space on laying hen feeding behavior and production performance in enriched colony housing.

PubMed

Oliveira, J L; Xin, H; Wu, H

2018-05-30

Current feeder space recommendations in laying hen welfare guidelines are inconsistent among and within countries. One determining criterion forming the recommendations (e.g. 12.0 cm/hen for the EU guideline) is that all birds can feed simultaneously. However, if there are other resources in the environment, as in enriched colony housing (ECH), it is unknown whether group-housed hens will choose to feed simultaneously. This study assesses the impact of feeder space on feeding behavior of 60 laying hens (W-36) in ECH using a ultra-high frequency radio-frequency identification-based tracking system. The feeder spaces investigated were 12.0, 9.5, 8.5 and 6.5 cm/hen, achieved by blocking portions of the overall feeder access to keep hens at the same stocking density. Each feeder space treatment, randomly assigned over the course of the experiment, lasted for 7 consecutive days. Feeding behaviors were characterized as daily time spent at the feeder (TS, min/hen-day), daily frequency of visits to the feeder (FV, #/hen-day), and maximum or average percentage of hens feeding simultaneously (MPB, APB, %). Group-average daily feed intake (FI, g/hen-day), water use (WU, g/hen-day), and hen-day egg production (HDEP, %) were also measured. The results revealed that at 12.0 cm/hen, where unoccupied feeder space was present, a maximum of 59.0±1.4% (average of 31.7±0.3%) hens fed simultaneously. No significant differences were detected among 12.0, 9.5 and 8.5 cm/hen in TS (293±10, 286±10 and 281±10 min/hen-day) and MPB (59.0±1.4, 57.3±1.4 and 53.3±1.4%) (P>0.05). The outcome of no significant differences also held true between 12.0 and 9.5 cm/hen in APB (31.7±0.3 v. 30.8±0.3%) and between 9.5 and 8.5 cm/hen in all response variables measured (P>0.05). However, there were significant differences in APB between 6.5 cm/hen and all other treatments; in TS and FV between 6.5 and 9.5 cm/hen; and in MPB between 6.5 and 12 cm/hen (P0.05). The results revealed that synchronous feeding of hens in the ECH did not increase with increasing feeder space. However, it is worth noting that lower feeder space may lead to aggression or frustration which was not quantified in the current study.

Accuracy of software-assisted detection of tumour feeders in transcatheter hepatic chemoembolization using three target definition protocols.

PubMed

Iwazawa, J; Ohue, S; Hashimoto, N; Mitani, T

2014-02-01

To compare the accuracy of computer software analysis using three different target-definition protocols to detect tumour feeder vessels for transarterial chemoembolization of hepatocellular carcinoma. C-arm computed tomography (CT) data were analysed for 81 tumours from 57 patients who had undergone chemoembolization using software-assisted detection of tumour feeders. Small, medium, and large-sized targets were manually defined for each tumour. The tumour feeder was verified when the target tumour was enhanced on selective C-arm CT of the investigated vessel during chemoembolization. The sensitivity, specificity, and accuracy of the three protocols were evaluated and compared. One hundred and eight feeder vessels supplying 81 lesions were detected. The sensitivity of the small, medium, and large target protocols was 79.8%, 91.7%, and 96.3%, respectively; specificity was 95%, 88%, and 50%, respectively; and accuracy was 87.5%, 89.9%, and 74%, respectively. The sensitivity was significantly higher for the medium (p = 0.003) and large (p < 0.001) target protocols than for the small target protocol. The specificity and accuracy were higher for the small (p < 0.001 and p < 0.001, respectively) and medium (p < 0.001 and p < 0.001, respectively) target protocols than for the large target protocol. The overall accuracy of software-assisted automated feeder analysis in transarterial chemoembolization for hepatocellular carcinoma is affected by the target definition size. A large target definition increases sensitivity and decreases specificity in detecting tumour feeders. A target size equivalent to the tumour size most accurately predicts tumour feeders. Copyright © 2013 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Foraging scent marks of bumblebees: footprint cues rather than pheromone signals

NASA Astrophysics Data System (ADS)

Wilms, Jessica; Eltz, Thomas

2008-02-01

In their natural habitat foraging bumblebees refuse to land on and probe flowers that have been recently visited (and depleted) by themselves, conspecifics or other bees, which increases their overall rate of nectar intake. This avoidance is often based on recognition of scent marks deposited by previous visitors. While the term ‘scent mark’ implies active labelling, it is an open question whether the repellent chemicals are pheromones actively and specifically released during flower visits, or mere footprints deposited unspecifically wherever bees walk. To distinguish between the two possibilities, we presented worker bumblebees ( Bombus terrestris) with three types of feeders in a laboratory experiment: unvisited control feeders, passive feeders with a corolla that the bee had walked over on its way from the nest (with unspecific footprints), and active feeders, which the bee had just visited and depleted, but which were immediately refilled with sugar water (potentially with specific scent marks). Bumblebees rejected both active and passive feeders more frequently than unvisited controls. The rate of rejection of passive feeders was only slightly lower than that of active feeders, and this difference vanished completely when passive corollas were walked over repeatedly on the way from the nest. Thus, mere footprints were sufficient to emulate the repellent effect of an actual feeder visit. In confirmation, glass slides on which bumblebees had walked on near the nest entrance accumulated hydrocarbons (alkanes and alkenes, C23 to C31), which had previously been shown to elicit repellency in flower choice experiments. We conclude that repellent scent marks are mere footprints, which foraging bees avoid when they encounter them in a foraging context.

Bacterial populations and adaptations in the mucus layers on living corals

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

Ducklow, H.W.; Mitchell, R.

1979-07-01

The external mucus layers of the stony coral Porites astreoides and the soft corals Palythoa sp. and Heteroxenia fuscesens are inhabited by communities of marine heterotrophic bacteria. Population levels of bacteria in coral mucus may be regulated by the self-cleaning behavior of the host. Bacterial populations in coral mucus respond to stresses applied to the host coral by growing to higher population levels in the mucus, indicating that these are populations of viable organisms closely attuned to host metabolism. Members of these microbial populations utilize the mucus compounds and may play a role in processing coral mucus for reef detritusmore » feeders. One such species, Vibrio alginolyticus, grows rapidly on Heteroxenia mucus, is attracted to dissolved mucus, and possesses a mechanism to maintain itself on the coral surface.« less

Voltage-Load Sensitivity Matrix Based Demand Response for Voltage Control in High Solar Penetration Distribution Feeders

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

Zhu, Xiangqi; Wang, Jiyu; Mulcahy, David

This paper presents a voltage-load sensitivity matrix (VLSM) based voltage control method to deploy demand response resources for controlling voltage in high solar penetration distribution feeders. The IEEE 123-bus system in OpenDSS is used for testing the performance of the preliminary VLSM-based voltage control approach. A load disaggregation process is applied to disaggregate the total load profile at the feeder head to each load nodes along the feeder so that loads are modeled at residential house level. Measured solar generation profiles are used in the simulation to model the impact of solar power on distribution feeder voltage profiles. Different casemore » studies involving various PV penetration levels and installation locations have been performed. Simulation results show that the VLSM algorithm performance meets the voltage control requirements and is an effective voltage control strategy.« less

Broadcasting satellite feeder links - Characteristics and planning

NASA Technical Reports Server (NTRS)

Kiebler, J. W.

1982-01-01

The paper presents the results of recent studies by the Feeder Link Sub-Working Group of the FCC Advisory Committee for the 1983 Regional Administrative Radio Conference (RARC). These studies conclude that specification of a few key parameters will make feeder link planning relatively straightforward. Feeder links can be located anywhere within a country if satellite orbit locations are separated by 10 deg for adjacent service areas and key parameter values presented in the paper are adopted. Colocated satellites serving a common service area need special attention to attain sufficient isolation between a desired channel and its adjacent cross-polarized channels and alternate co-polarized channels. In addition to presenting planning conclusions by the Advisory Committee, the paper presents and analyzes actions of the International Radio Consultative Committee's Conference Planning Meeting (CPM) concerning feeder links. The CPM reached conclusions similar to, and compatible with, those of the Advisory Committee.

Consequences of Feeder Delays for the Success of A380 Operations

NASA Technical Reports Server (NTRS)

Ruehle, Jens; Goetsch, Bjoern; Koch, Benjamin

2006-01-01

Due to existing slot and infrastructure constraints at international hub-and-spoke airports, an increase in feeder traffic seems only possible if larger feeder aircraft are used. Using a case study of Lufthansa German Airlines at Frankfurt International Airport, three possible A380 routes (Beijing, Tokyo-Narita, Los Angeles) were examined to assess the extent to which delays of feeder traffic may impact the economic performance of very large aircraft. On the basis of today s delays and anticipated traffic growth in the future, we found that between 9.5% and 13.5% of connecting passengers are unable to transfer to their respective intercontinental flights. In addition, the results demonstrate that a further increase in delays can be detrimental to the profitable operation of very large aircraft, as demonstrated by two out of three simulated routes. We suggest options for airlines operating very large aircraft to counteract the negative impacts of feeder delays.

Effects of bale feeder type and supplementation of monensin on hay waste, intake, and performance of beef cattle

USDA-ARS?s Scientific Manuscript database

The effects of feeder type and supplemental monensin on hay utilization in beef cows was investigated using 56 crossbred beef cows (BW= 494 ± 50 kg; BCS= 5.2 ± 0.5) in a split-plot treatment arrangement with a completely randomized design. Supplement treatment served as the main plot and feeder desi...

High pressure rotary piston coal feeder

NASA Technical Reports Server (NTRS)

Gardner, J. F.; Gencsoy, H. T.; Strimbeck, D. C.

1977-01-01

This feeder concept uniquely combines the functions of solids feeding, metering, and pressurization into one compact system. Success with the rotary-piston concept would provide a lower-cost alternative to lock-hopper systems. The design of the feeder is presented, with special emphasis on the difficult problem of seal design. Initial tests will be to check seal performance. Subsequent tests will evaluate solids-feeding ability.

Low Spillage Metabolic Feeder

NASA Technical Reports Server (NTRS)

Evans, JuliAnn (Inventor); Gundo, Daniel P. (Inventor); Harper, Jennifer S. (Inventor); Mulenburg, Gerald M. (Inventor); Skundberg, Thomas L. (Inventor)

1996-01-01

An animal feeder for use in a metabolic cage is introduced. The feeder includes a confined passageway and an adjustable notched gate proceeding a food cup. The gate is adjusted so that the entry area to the food cup approximates the cross sectional head area of the animal. Food ejected from the food cup by a caged animal is dropped through a grate into a spill tray.

7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 10 2011-01-01 2011-01-01 false Collection and remittance of assessments for the sale of feeder pigs and market hogs. 1230.113 Section 1230.113 Agriculture Regulations of the Department... pigs and market hogs. Pursuant to the provisions of § 1230.71, purchasers of feeder pigs or market hogs...

7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 10 2014-01-01 2014-01-01 false Collection and remittance of assessments for the sale of feeder pigs and market hogs. 1230.113 Section 1230.113 Agriculture Regulations of the Department... pigs and market hogs. Pursuant to the provisions of § 1230.71, purchasers of feeder pigs or market hogs...

7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 10 2012-01-01 2012-01-01 false Collection and remittance of assessments for the sale of feeder pigs and market hogs. 1230.113 Section 1230.113 Agriculture Regulations of the Department... pigs and market hogs. Pursuant to the provisions of § 1230.71, purchasers of feeder pigs or market hogs...

7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 10 2013-01-01 2013-01-01 false Collection and remittance of assessments for the sale of feeder pigs and market hogs. 1230.113 Section 1230.113 Agriculture Regulations of the Department... pigs and market hogs. Pursuant to the provisions of § 1230.71, purchasers of feeder pigs or market hogs...

7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Collection and remittance of assessments for the sale of feeder pigs and market hogs. 1230.113 Section 1230.113 Agriculture Regulations of the Department... pigs and market hogs. Pursuant to the provisions of § 1230.71, purchasers of feeder pigs or market hogs...

Assessing the role of benthic filter feeders on phytoplankton production in a shellfish farming site: Mont Saint Michel Bay, France

NASA Astrophysics Data System (ADS)

Cugier, Philippe; Struski, Caroline; Blanchard, Michel; Mazurié, Joseph; Pouvreau, Stéphane; Olivier, Frédéric; Trigui, Jihane R.; Thiébaut, Eric

2010-07-01

The macrobenthic community of Mont Saint Michel Bay (English Channel, France) is mainly dominated by filter feeders, including cultivated species (oysters and mussels). An ecological model of the bay was developed, coupling a 2D hydro-sedimentary model and two biological models for primary production and filter-feeder filtration. The filter-feeder model includes three cultivated species ( Mytilus edulis, Crassostrea gigas and Ostrea edulis), one invasive species ( Crepidula fornicata) and eight wild native species ( Abra alba, Cerastoderma edule, Glycymeris glycymeris, Lanice conchilega, Macoma balthica, Paphia rhomboides, Sabellaria alveolata, andSpisula ovalis). For cultivated and invasive species, the production of biodeposits was computed to assess their role in restimulating primary production. Chlorophyll a concentrations appeared to be strongly controlled by the filter feeders. When the pressure of each benthic compartment on phytoplankton was estimated separately wild species and the invasive slipper limpet C.fornicata were shown to be key elements in the control of primary production. Conversely, the role of cultivated species, particularly oysters, was weaker. Feedback due to the mineralization of biodeposits also appears to be crucial to fully evaluate the role of filter feeders in primary production.

High precision during food recruitment of experienced (reactivated) foragers in the stingless bee Scaptotrigona mexicana (Apidae, Meliponini)

NASA Astrophysics Data System (ADS)

Sánchez, Daniel; Nieh, James C.; Hénaut, Yann; Cruz, Leopoldo; Vandame, Rémy

Several studies have examined the existence of recruitment communication mechanisms in stingless bees. However, the spatial accuracy of location-specific recruitment has not been examined. Moreover, the location-specific recruitment of reactivated foragers, i.e., foragers that have previously experienced the same food source at a different location and time, has not been explicitly examined. However, such foragers may also play a significant role in colony foraging, particularly in small colonies. Here we report that reactivated Scaptotrigona mexicana foragers can recruit with high precision to a specific food location. The recruitment precision of reactivated foragers was evaluated by placing control feeders to the left and the right of the training feeder (direction-precision tests) and between the nest and the training feeder and beyond it (distance-precision tests). Reactivated foragers arrived at the correct location with high precision: 98.44% arrived at the training feeder in the direction trials (five-feeder fan-shaped array, accuracy of at least +/-6° of azimuth at 50 m from the nest), and 88.62% arrived at the training feeder in the distance trials (five-feeder linear array, accuracy of at least +/-5 m or +/-10% at 50 m from the nest). Thus, S. mexicana reactivated foragers can find the indicated food source at a specific distance and direction with high precision, higher than that shown by honeybees, Apis mellifera, which do not communicate food location at such close distances to the nest.

Number of hummingbird visits determines flower mite abundance on hummingbird feeders.

PubMed

Márquez-Luna, Ubaldo; Vázquez González, María Magdalena; Castellanos, Ignacio; Ortiz-Pulido, Raúl

2016-08-01

Members of several genera of mites from the family Melicharidae (Mesostigmata) use hummingbirds as transport host to move from flower to flower, where they feed on pollen and nectar. The factors that influence hummingbird flower mite abundance on host plant flowers are not currently known. Here we tested whether hummingbird flower mite abundance on an artificial nectar source is determined by number of hummingbird visits, nectar energy content or species richness of visiting hummingbirds. We conducted experiments employing hummingbird feeders with sucrose solutions of low, medium, and high energy concentrations, placed in a xeric shrubland. In the first experiment, we recorded the number of visiting hummingbirds and the number of visiting hummingbird species, as well as the abundance of hummingbird flower mites on each feeder. Feeders with the highest sucrose concentration had the most hummingbird visits and the highest flower mite abundances; however, there was no significant effect of hummingbird species richness on mite abundance. In the second experiment, we recorded flower mite abundance on feeders after we standardized the number of hummingbird visits to them. Abundance of flower mites did not differ significantly between feeders when we controlled for hummingbird visits. Our results suggest that nectar energy concentration determines hummingbird visits, which in turn determines flower mite abundance in our feeders. Our results do not support the hypothesis that mites descend from hummingbird nostrils more on richer nectar sources; however, it does not preclude the possibility that flower mites select for nectar concentration at other spatial and temporal scales.

Prolonged survival of reconstituted skin grafts without immunosuppression.

PubMed

Sasamoto, Y; Alexander, J W; Babcock, G F

1990-01-01

Reconstituted skin composed of a cultured allogeneic epithelial sheet (CAES) and a cultured allogeneic dermis (CAD) was evaluated in a rat model to determine whether it could survive for a prolonged period without immunosuppression. Additionally, free CAD grafts were evaluated for their suitability as dermal substitutes. Male Buffalo rats were used as donors and male Lewis rats as recipients. Split-thickness skin obtained from Buffalo rats was separated into epidermis and dermis by means of Dispase II enzyme. The epidermal layers were minced and trypsinized. Then dispersed single keratinocytes were inoculated onto a irradiated 3T3 cell feeder layer. After a suitable period, a confluent cultured keratinocyte layer was detached and provided CAES grafts. Cultured allogeneic dermis grafts were prepared from cultures of the dermal component. Cultured allogeneic dermis grafts, covered by split thickness isografts (STIG) or local skin flaps, became revascularized at a rate of 94.6% and 90.9%, respectively, 7 days after grafting. However, only 25% of CAD grafts covered by synthetic materials became vascularized. Four types of wound coverage were compared including: (1) CAES grafts, (2) CAES over CAD grafts, (3) split-thickness isografts, and (4) STIG over CAD grafts. In groups 2 and 4, CAD grafts were applied 7 days before CAES grafts or STIG. Grafts of groups 1 and 2 were successful in only 36.7% and 31.1% of the animals and resulted in a high rate of wound contracture--72.4%, 66.7%, respectively. On the other hand, in groups 3 and 4, higher average rates of revascularization (92.0% and 88.3%) and lower rates of wound contracture (25.4% and 24.2%) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of Pratylenchus penetrans and Rhizoctonia fragariae in Strawberry Black Root Rot

PubMed Central

LaMondia, J. A.

2003-01-01

A split-root technique was used to examine the interaction between Pratylenchus penetrans and the cortical root-rotting pathogen Rhizoctonia fragariae in strawberry black root rot. Plants inoculated with both pathogens on the same half of a split-root crown had greater levels of root rot than plants inoculated separately or with either pathogen alone. Isolation of R. fragariae from field-grown roots differed with root type and time of sampling. Fungal infection of structural roots was low until fruiting, whereas perennial root colonization was high. Isolation of R. fragariae from feeder roots was variable, but was greater from feeder roots on perennial than from structural roots. Isolation of the fungus was greater from structural roots with nematode lesions than from non-symptomatic roots. Rhizoctonia fragariae was a common resident on the sloughed cortex of healthy perennial roots. From this source, the fungus may infect additional roots. The direct effects of lesion nematode feeding and movement are cortical cell damage and death. Indirect effects include discoloration of the endodermis and early polyderm formation. Perhaps weakened or dying cells caused directly or indirectly by P. penetrans are more susceptible to R. fragariae, leading to increased disease. PMID:19265969

Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

PubMed

Wang, Juan; Hao, Jie; Bai, Donghui; Gu, Qi; Han, Weifang; Wang, Lei; Tan, Yuanqing; Li, Xia; Xue, Ke; Han, Pencheng; Liu, Zhengxin; Jia, Yundan; Wu, Jun; Liu, Lei; Wang, Liu; Li, Wei; Liu, Zhonghua; Zhou, Qi

2015-11-12

Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.

47 CFR 25.258 - Sharing between NGSO MSS Feeder links Stations and GSO FSS services in the 29.25-29.5 GHz Bands.

Code of Federal Regulations, 2012 CFR

2012-10-01

.... (a) Operators of NGSO MSS feeder link earth stations and GSO FSS earth stations in the band 29.25 to... MSS feeder link earth station complexes, that will minimize instances of unacceptable interference to the GSO FSS space stations. Earth station licensees operating with GSO FSS systems shall be capable of...

47 CFR 25.257 - Special requirements for operations in the band 29.1-29.25 GHz between NGSO MSS and LMDS.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Earth-to-space transmissions from feeder link earth station complexes. A “feeder link earth station complex” may include up to three (3) earth station groups, with each earth station group having up to four... NGSO MSS licensees or applicants pursuant to § 101.147. (b) A maximum of seven (7) feeder link earth...

47 CFR 25.257 - Special requirements for operations in the band 29.1-29.25 GHz between NGSO MSS and LMDS.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Earth-to-space transmissions from feeder link earth station complexes. A “feeder link earth station complex” may include up to three (3) earth station groups, with each earth station group having up to four... NGSO MSS licensees or applicants pursuant to § 101.147. (b) A maximum of seven (7) feeder link earth...

47 CFR 25.257 - Special requirements for operations in the band 29.1-29.25 GHz between NGSO MSS and LMDS.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Earth-to-space transmissions from feeder link earth station complexes. A “feeder link earth station complex” may include up to three (3) earth station groups, with each earth station group having up to four... NGSO MSS licensees or applicants pursuant to § 101.147. (b) A maximum of seven (7) feeder link earth...

47 CFR 25.257 - Special requirements for operations in the band 29.1-29.25 GHz between NGSO MSS and LMDS.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Earth-to-space transmissions from feeder link earth station complexes. A “feeder link earth station complex” may include up to three (3) earth station groups, with each earth station group having up to four... NGSO MSS licensees or applicants pursuant to § 101.147. (b) A maximum of seven (7) feeder link earth...

47 CFR 25.258 - Sharing between NGSO MSS Feeder links Stations and GSO FSS services in the 29.25-29.5 GHz Bands.

Code of Federal Regulations, 2011 CFR

2011-10-01

.... (a) Operators of NGSO MSS feeder link earth stations and GSO FSS earth stations in the band 29.25 to... MSS feeder link earth station complexes, that will minimize instances of unacceptable interference to the GSO FSS space stations. Earth station licensees operating with GSO FSS systems shall be capable of...

47 CFR 25.258 - Sharing between NGSO MSS Feeder links Stations and GSO FSS services in the 29.25-29.5 GHz Bands.

Code of Federal Regulations, 2013 CFR

2013-10-01

.... (a) Operators of NGSO MSS feeder link earth stations and GSO FSS earth stations in the band 29.25 to... MSS feeder link earth station complexes, that will minimize instances of unacceptable interference to the GSO FSS space stations. Earth station licensees operating with GSO FSS systems shall be capable of...

47 CFR 25.223 - Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS.

Code of Federal Regulations, 2013 CFR

2013-10-01

... feeder link earth stations in the 17/24 GHz BSS. 25.223 Section 25.223 Telecommunication FEDERAL....223 Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS. (a) This section applies to all applications for earth station licenses in the 17/24 GHz BSS frequency...

47 CFR 25.223 - Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS.

Code of Federal Regulations, 2011 CFR

2011-10-01

... feeder link earth stations in the 17/24 GHz BSS. 25.223 Section 25.223 Telecommunication FEDERAL....223 Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS. (a) This section applies to all applications for earth station licenses in the 17/24 GHz BSS frequency...

47 CFR 25.258 - Sharing between NGSO MSS Feeder links Stations and GSO FSS services in the 29.25-29.5 GHz Bands.

Code of Federal Regulations, 2010 CFR

2010-10-01

.... (a) Operators of NGSO MSS feeder link earth stations and GSO FSS earth stations in the band 29.25 to... MSS feeder link earth station complexes, that will minimize instances of unacceptable interference to the GSO FSS space stations. Earth station licensees operating with GSO FSS systems shall be capable of...

47 CFR 25.223 - Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS.

Code of Federal Regulations, 2012 CFR

2012-10-01

... feeder link earth stations in the 17/24 GHz BSS. 25.223 Section 25.223 Telecommunication FEDERAL....223 Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS. (a) This section applies to all applications for earth station licenses in the 17/24 GHz BSS frequency...

47 CFR 25.258 - Sharing between NGSO MSS Feeder links Stations and GSO FSS services in the 29.25-29.5 GHz Bands.

Code of Federal Regulations, 2014 CFR

2014-10-01

.... (a) Operators of NGSO MSS feeder link earth stations and GSO FSS earth stations in the band 29.25 to... MSS feeder link earth station complexes, that will minimize instances of unacceptable interference to the GSO FSS space stations. Earth station licensees operating with GSO FSS systems shall be capable of...

47 CFR 25.257 - Special requirements for operations in the band 29.1-29.25 GHz between NGSO MSS and LMDS.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Earth-to-space transmissions from feeder link earth station complexes. A “feeder link earth station complex” may include up to three (3) earth station groups, with each earth station group having up to four... NGSO MSS licensees or applicants pursuant to § 101.147. (b) A maximum of seven (7) feeder link earth...

47 CFR 25.223 - Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS.

Code of Federal Regulations, 2010 CFR

2010-10-01

... feeder link earth stations in the 17/24 GHz BSS. 25.223 Section 25.223 Telecommunication FEDERAL....223 Off-axis EIRP spectral density limits for feeder link earth stations in the 17/24 GHz BSS. (a) This section applies to all applications for earth station licenses in the 17/24 GHz BSS frequency...

The Effects of a Modified Treatment Package with and without Feeder Modeling on One Child's Acceptance of Novel Foods

ERIC Educational Resources Information Center

Seiverling, Laura; Harclerode, Whitney; Williams, Keith

2014-01-01

The purpose of this study was to examine if sequential presentation with feeder modeling would lead to an increase in bites accepted of new foods compared to sequential presentation without feeder modeling in a typically developing 4-year-old boy with food selectivity. The participant's acceptance of novel foods increased both in the modeling and…

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

PubMed

Bukong, Terence N; Lo, Tracie; Szabo, Gyongyi; Dolganiuc, Angela

2012-05-01

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation. © 2012 John Wiley & Sons A/S.

Wean-to-finish feeder space availability effects on nursery and finishing pig performance and total tract digestibility in a commercial setting when feeding dried distillers grains with solubles.

PubMed

Weber, E K; Stalder, K J; Patience, J F

2015-04-01

The study objectives were to determine nursery phase feeder space allowance effects on pig performance when double stocked and, second, to determine feeder space allowance and dried distillers grains with solubles (DDGS) inclusion level effects on pig performance and nutrient digestibility during the growing-finishing phase. This study was performed on the same group of pigs within a commercial wean-finish system. For the nursery phase, a completely randomized design was used to compare 3 feeder space allowance treatments (2.1, 2.5, and 2.9 cm/pig). A total of 3,720 pigs were randomly allotted to same-sex pens (10 feeders/treatment) housing 62 pigs/pen. Thirty 7-hole, double-sided feeders were utilized in the study. Differing linear feeder space treatments were established by blocking off sections for the nursery and grow-finish portions of this experiment. All pigs were provided equal floor space (0.26 m2/pig). In the grow-finish phase, a total of 1,860 pigs (n = 60 pens) were utilized in a 2 × 3 factorial design with 3 feeder space allowances (4.1, 4.9, or 5.7 cm/pig) and 2 dietary DDGS treatments (30% [D30] or 60% [D60]). Fecal and diet samples were collected and analyzed to estimate apparent total tract digestibility percentage (ATTD %). In the nursery portion of the trial, there was no feeder space treatment effect on ADG, ADFI, or feed efficiency (P > 0.10) from weaning to d 56 postweaning or during any weigh period. In the grow-finish portion of the trial, feeder space allowance and DDGS inclusion level did not affect ADG, ADFI, or feed efficiency (P > 0.05) from d 57 postweaning to market. Pigs fed the D30 diet had greater HCW, percent yield, and loin depth than those on the D60 diet (P < 0.05). Pigs fed the D30 treatment had greater (P < 0.05) ATTD for DM and GE for both collection periods compared with those on the D60 treatment. In summary, feeder space allowance did not impact pig performance during the nursery or grow-finish production phases. Inclusion of DDGS at higher levels will decrease ADFI but not ADG or efficiency when isocaloric diets are fed. The inclusion level of DDGS does impact HCW and percent yield because of increasing intestinal weights when pigs are fed diets containing increasing DDGS inclusion rates. Dry matter and energy digestibility were greater in pigs fed the lower DDGS treatment.

Effect of concentrate feeder design on performance, eating and animal behavior, welfare, ruminal health, and carcass quality in Holstein bulls fed high-concentrate diets.

PubMed

Verdú, M; Bach, A; Devant, M

2015-06-01

A total of 240 Holstein bulls (121 ± 2.0 kg initial BW; 99 ± 1.0 d of age), from 2 consecutive fattening cycles, were randomly allocated in 1 of 6 pens and assigned to 1 of the 3 treatments consisting of different concentrate feeder designs: a control feeder with 4 feeding spaces (CF), a feeder with less concentrate capacity (CFL), and a single-space feeder with lateral protections (SF). Each pen had a straw feeder and a drinker. All animals were fed a high-concentrate diet for ad libitum intake. Concentrate consumption was recorded daily using a computerized feeder, straw consumption was recorded weekly, and BW was recorded every 14 d. Animal behavior was registered on d 1, 3, 5, 8, and 14 and every 28 d by scan sampling. Eating behavior at concentrate feeders was filmed on d 12, 125, and 206. On d 7, 120, and 204, samples of rumen contents were collected for measurement of pH and VFA and blood samples were obtained to analyze NEFA, haptoglobin, glucose, and insulin. Animals were slaughtered after 223 d, and HCW and lesions of the rumen wall and liver were recorded. The accumulative concentrate consumption per animal tended (P = 0.09) to be greater with CF than with CFL and SF. Also, CV of concentrate consumption was greater (P < 0.01) for SF than for CF or CFL. However, feeder design did not influence the other performance and carcass data. Also, no differences among treatments in rumen wall evaluation and liver abscesses were observed. At 7 and 204 d of study, SF bulls had greater (P < 0.05) rumen pH compared with CF and CFL bulls. On d 7, the acetate to propionate ratio from SF was greater (P < 0.05) than for CFL or CF. At d 7, NEFA of SF were greater (P < 0.05) compared with CF and CFL. Bulls fed with CF have the greatest (P < 0.01) concentrate disappearance velocity followed by bulls fed with CFL and finally by bulls fed with SF, and this was associated with different feeding behaviors. Bulls on SF spent more time (P < 0.05) eating straw and exhibited fewer (P < 0.05) displacements at concentrate feeder than CF and CFL bulls. The CFL bulls exhibited (P < 0.01) more attempted mounts and tended (P = 0.10) to exhibit more completed mounts than CF bulls. In conclusion, both alternative feeder designs (CFL and SF) are good strategies to reduce total concentrate consumption without impairing performance, rumen health, or animal welfare in Holstein bulls fed high-concentrate diets. However, at the beginning, there was evidence that animals fed using SF had problems with adaptation.

Effect of feeder space during the growing and laying periods and the rate of feed increase at the onset of lay on broiler breeder female reproductive function.

PubMed

Leksrisompong, N; Romero-Sanchez, H; Oviedo-Rondón, E O; Brake, J

2014-07-01

A study was conducted to examine how 2 feeder space allocations during the rearing period followed by 2 feeder space allocations after photostimulation and 2 female feeding to peak programs (fast or slow) affected female broiler breeder reproductive performance and mortality. Sixteen pens of 76 breeder females each were equipped with either 4 tube feeders with a 132 cm circumference pan (7.0 cm/female) or 6 feeders (10.4 cm/female) to 21 wk of age. Thereafter, 64 females were moved to breeding pens, photostimulated, and fed sex-separate from either 3 (6.2 cm/female) or 5 (10.3 cm/female) feeders with either fast or slow feeding to peak feeding programs applied to complete a 2 × 2 × 2 factorial design. Seven males that were separately reared in a similar manner were added per pen. Individual female BW was determined at 6, 20, and 32 wk of age and BW uniformity assessed. Greater feeder space during rearing increased BW at 32 wk of age, whereas greater feeder space during lay or slow feeding to peak decreased BW at 32 wk. There were no differences in BW uniformity. Hens from the 10.4 to 10.3 cm/female combination produced a significantly greater number of eggs as compared with the 7.0 to 10.3 cm/female and 10.4 to 6.2 cm/female combinations with the 7.0 to 6.2 cm/female combination intermediate. Percentage hen-day egg production of the 10.4 to 10.3 cm/female combination hens was significantly greater than all other combinations. Livability was improved in the 10.4 to 10.3 cm/female combination relative to the 7.0 to 10.3 cm/female combination with the others intermediate. The fast feeding to peak program increased yolk weight as well as yolk:albumen ratio at 28 and 30 wk of age, but egg weight did not differ. These data indicated that increased or decreased feeder space between the growing and laying periods did not affect broiler breeder female BW, uniformity, egg weight, fertility, or hatchability. The 10.3 cm/female laying feeder space exhibited the best hen-day egg production in combination with 10.4 cm/pullet rearing but not with 7.0 cm/pullet rearing space. In a similar manner, hen mortality was greater in the 7.0 to 10.3 cm/female feeder space combination that the 10.4 to 10.3 cm/female combination. © 2014 Poultry Science Association Inc.

IL-33, IL-25, and TSLP induce a distinct phenotypic and activation profile in human type 2 innate lymphoid cells.

PubMed

Camelo, Ana; Rosignoli, Guglielmo; Ohne, Yoichiro; Stewart, Ross A; Overed-Sayer, Catherine; Sleeman, Matthew A; May, Richard D

2017-04-11

Innate lymphoid cells (ILCs) represent a distinct branch of the lymphoid lineage composed of 3 major subpopulations: ILC1, ILC2, and ILC3. ILCs are mainly described as tissue-resident cells but can be detected at low levels in human blood. However, unlike mouse ILCs, there is still no consistent methodology to purify and culture these cells that enables in-depth analysis of their intrinsic biology. Here, we describe defined culture conditions for ILC2s, which allowed us to dissect the roles of interleukin 2 (IL-2), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) individually, or in combination, in modulating ILC2 phenotype and function. We show that TSLP is important for ILC2 survival, while ILC2 activation is more dependent on IL-33, especially when in combination with IL-2 or TSLP. We found that activation of ILC2s by IL-33 and TSLP dramatically upregulated their surface expression of c-Kit and downregulated expression of the canonical markers IL-7Rα and CRTH2. IL-2 further amplified ILC2 production of IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor but also induced a more natural killer (NK)-like phenotype in ILC2, with upregulation of granzyme B production by these cells. Furthermore, ILC2 plasticity was observed in serum-free SFEM II media in response to IL-33, IL-25, and TSLP stimulation and independently of IL-12 and IL-1β. This is the first comprehensive report of an in vitro culture system for human ILC2s, without the use of feeder layers, which additionally evaluates the impact of IL-25, IL-33, and TSLP alone or in combination on ILC2 surface phenotype and activation status.

Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

PubMed Central

Li, W; Chen, Y-T; Hayashida, Y; Blanco, G; Kheirkah, A; He, H; Chen, S-Y; Liu, C-Y; Tseng, SCG

2010-01-01

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens–Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP–Pax6 transgenes into these Pax6− clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium. PMID:18027901

Bivalve grazing can shape phytoplankton communities

USGS Publications Warehouse

Lucas, Lisa; Cloern, James E.; Thompson, Janet K.; Stacey, Mark T.; Koseff, Jeffrey K.

2016-01-01

The ability of bivalve filter feeders to limit phytoplankton biomass in shallow waters is well-documented, but the role of bivalves in shaping phytoplankton communities is not. The coupled effect of bivalve grazing at the sediment-water interface and sinking of phytoplankton cells to that bottom filtration zone could influence the relative biomass of sinking (diatoms) and non-sinking phytoplankton. Simulations with a pseudo-2D numerical model showed that benthic filter feeding can interact with sinking to alter diatom:non-diatom ratios. Cases with the smallest proportion of diatom biomass were those with the fastest sinking speeds and strongest bivalve grazing rates. Hydrodynamics modulated the coupled sinking-grazing influence on phytoplankton communities. For example, in simulations with persistent stratification, the non-sinking forms accumulated in the surface layer away from bottom grazers while the sinking forms dropped out of the surface layer toward bottom grazers. Tidal-scale stratification also influenced vertical gradients of the two groups in opposite ways. The model was applied to Suisun Bay, a low-salinity habitat of the San Francisco Bay system that was transformed by the introduction of the exotic clam Potamocorbula amurensis. Simulation results for this Bay were similar to (but more muted than) those for generic habitats, indicating that P. amurensis grazing could have caused a disproportionate loss of diatoms after its introduction. Our model simulations suggest bivalve grazing affects both phytoplankton biomass and community composition in shallow waters. We view these results as hypotheses to be tested with experiments and more complex modeling approaches.

Three-dimensional model of an ultramafic feeder system to the Nikolai Greenstone mafic large igneous province, central Alaska Range

USGS Publications Warehouse

Glen, J.M.G.; Schmidt, J.M.; Connard, G.G.

2011-01-01

The Amphitheater Mountains and southern central Alaska Range expose a thick sequence of Triassic Nikolai basalts that is underlain by several mafic-ultramafic complexes, the largest and best exposed being the Fish Lake and Tangle (FL-T) mafic-ultramafic sills that flank the Amphitheater Mountains synform. Three-dimensional (3-D) modeling of gravity and magnetic data reveals details of the structure of the Amphitheater Mountains, such as the orientation and thickness of Nikolai basalts, and the geometry of the FL-T intrusions. The 3-D model (50 ?? 70 km) includes the full geographic extent of the FL-T complexes and consists of 11 layers. Layer surfaces and properties (density and magnetic susceptibility) were modified by forward and inverse methods to reduce differences between the observed and calculated gravity and magnetic grids. The model suggests that the outcropping FL-T sills are apparently connected and traceable at depth and reveals variations in thickness, shape, and orientation of the ultramafic bodies that may identify paths of magma flow. The model shows that a significant volume (2000 km3) of ultramafic material occurs in the subsurface, gradually thickening and plunging westward to depths exceeding 4 km. This deep ultramafic material is interpreted as the top of a keel or root system that supplied magma to the Nikolai lavas and controlled emplacement of related magmatic intrusions. The presence of this deep, keel-like structure, and asymmetry of the synform, supports a sag basin model for development of the Amphitheater Mountains structure and reveals that the feeders to the Nikolai are much more extensive than previously known. Copyright 2011 by the American Geophysical Union.

Analysis of PV Advanced Inverter Functions and Setpoints under Time Series Simulation.

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

Seuss, John; Reno, Matthew J.; Broderick, Robert Joseph

Utilities are increasingly concerned about the potential negative impacts distributed PV may have on the operational integrity of their distribution feeders. Some have proposed novel methods for controlling a PV system's grid - tie inverter to mitigate poten tial PV - induced problems. This report investigates the effectiveness of several of these PV advanced inverter controls on improving distribution feeder operational metrics. The controls are simulated on a large PV system interconnected at several locations within two realistic distribution feeder models. Due to the time - domain nature of the advanced inverter controls, quasi - static time series simulations aremore » performed under one week of representative variable irradiance and load data for each feeder. A para metric study is performed on each control type to determine how well certain measurable network metrics improve as a function of the control parameters. This methodology is used to determine appropriate advanced inverter settings for each location on the f eeder and overall for any interconnection location on the feeder.« less

Analysis of Feeder Bus Network Design and Scheduling Problems

PubMed Central

Almasi, Mohammad Hadi; Karim, Mohamed Rehan

2014-01-01

A growing concern for public transit is its inability to shift passenger's mode from private to public transport. In order to overcome this problem, a more developed feeder bus network and matched schedules will play important roles. The present paper aims to review some of the studies performed on Feeder Bus Network Design and Scheduling Problem (FNDSP) based on three distinctive parts of the FNDSP setup, namely, problem description, problem characteristics, and solution approaches. The problems consist of different subproblems including data preparation, feeder bus network design, route generation, and feeder bus scheduling. Subsequently, descriptive analysis and classification of previous works are presented to highlight the main characteristics and solution methods. Finally, some of the issues and trends for future research are identified. This paper is targeted at dealing with the FNDSP to exhibit strategic and tactical goals and also contributes to the unification of the field which might be a useful complement to the few existing reviews. PMID:24526890

Arthropods associated with fungal galls: do large galls support more abundant and diverse inhabitants?

NASA Astrophysics Data System (ADS)

Funamoto, Daichi; Sugiura, Shinji

2017-02-01

Fungus-induced galls can attract spore-feeding arthropods as well as gall-feeding ones, resulting in diverse communities. Do large fungal galls support more abundant and diverse arthropod communities than small fungal galls? To address this question, we investigated the structure of the arthropod community associated with bud galls induced by the fungus Melanopsichium onumae on the tree species Cinnamomum yabunikkei (Lauraceae) in central Japan. Thirteen species of arthropods were associated with M. onumae galls. Dominant arthropod species were represented by the larvae of a salpingid beetle (a spore feeder), a nitidulid beetle (a spore feeder), a cosmopterigid moth (a spore feeder), an unidentified moth (a gall tissue feeder), and a drosophilid species (a gall tissue feeder). Arthropod abundance and species richness were positively correlated with gall diameter. The majority of the most abundant species were more frequently found in large galls than in small ones, indicating that large fungal galls, which have more food and/or space for arthropods, could support a more abundant and diverse arthropod community.

Social and nonsocial behaviours of sex- and age-matched enclosed and free-ranging rhesus monkeys (Macaca mulatta).

PubMed

Baulu, J; Redmond, D E

1980-01-01

The behavioural profiles (time budgeting of social and nonsocial activities) and the frequencies of major social interactions of corral-enclosed rhesus monkeys were compared with sex- and age-matched free-ranging rhesus monkeys on La Cueva Island, Puerto Rico. All animals (n = 32) were provisioned ad libitum at specific feeder sites. The occurrence of 14 behaviours around feeders was compared with their occurrence away from the feeders by noting the location of each monkey relative to the feeder at the time of observation. An analysis of variance between free-ranging versus corral-enclosed groups and within groups by location (around or away from the feeder) revealed significant differences in several behavioural categories, including foraging, lookout, inactive, dominant, submissive, allogrooming, social contact, social initiative, active, and passive behaviours. When the effect of limited food distribution sites was analyzed by comparing data recorded away from the feeding sites, there were remarkably few differences between the groups.

A new angle on microscopic suspension feeders near boundaries.

PubMed

Pepper, Rachel E; Roper, Marcus; Ryu, Sangjin; Matsumoto, Nobuyoshi; Nagai, Moeto; Stone, Howard A

2013-10-15

Microscopic sessile suspension feeders are a critical component in aquatic ecosystems, acting as an intermediate trophic stage between bacteria and higher eukaryotic taxa. Because they live attached to boundaries, it has long been thought that recirculation of the feeding currents produced by sessile suspension feeders inhibits their ability to access fresh fluid. However, previous models for the feeding flows of these organisms assume that they feed by pushing fluid perpendicular to surfaces they live upon, whereas we observe that sessile suspension feeders often feed at an angle to these boundaries. Using experiments and calculations, we show that living suspension feeders (Vorticella) likely actively regulate the angle that they feed relative to a substratum. We then use theory and simulations to show that angled feeding increases nutrient and particle uptake by reducing the reprocessing of depleted water. This work resolves an open question of how a key class of suspension-feeding organisms escapes physical limitations associated with their sessile lifestyle. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nutrient compensatory foraging in a free-living social insect

NASA Astrophysics Data System (ADS)

Christensen, Keri L.; Gallacher, Anthony P.; Martin, Lizzie; Tong, Desmond; Elgar, Mark A.

2010-10-01

The geometric framework model predicts that animal foraging decisions are influenced by their dietary history, with animals targeting a combination of essential nutrients through compensatory foraging. We provide experimental confirmation of nutrient-specific compensatory foraging in a natural, free-living population of social insects by supplementing their diet with sources of protein- or carbohydrate-rich food. Colonies of the ant Iridomyrmex suchieri were provided with feeders containing food rich in either carbohydrate or protein for 6 days, and were then provided with a feeder containing the same or different diet. The patterns of recruitment were consistent with the geometric framework: while feeders with a carbohydrate diet typically attracted more workers than did feeders with protein diet, the difference in recruitment between the two nutrients was smaller if the colonies had had prior access to carbohydrate than protein. Further, fewer ants visited feeders if the colony had had prior access to protein than to carbohydrates, suggesting that the larvae play a role in worker foraging behaviour.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

PubMed Central

Foltz, Jennifer A.; Somanchi, Srinivas S.; Yang, Yanwen; Aquino-Lopez, Arianexys; Bishop, Erin E.; Lee, Dean A.

2016-01-01

Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy. PMID:27933061

An enhanced SOCP-based method for feeder load balancing using the multi-terminal soft open point in active distribution networks

DOE PAGES

Ji, Haoran; Wang, Chengshan; Li, Peng; ...

2017-09-20

The integration of distributed generators (DGs) exacerbates the feeder power flow fluctuation and load unbalanced condition in active distribution networks (ADNs). The unbalanced feeder load causes inefficient use of network assets and network congestion during system operation. The flexible interconnection based on the multi-terminal soft open point (SOP) significantly benefits the operation of ADNs. The multi-terminal SOP, which is a controllable power electronic device installed to replace the normally open point, provides accurate active and reactive power flow control to enable the flexible connection of feeders. An enhanced SOCP-based method for feeder load balancing using the multi-terminal SOP is proposedmore » in this paper. Furthermore, by regulating the operation of the multi-terminal SOP, the proposed method can mitigate the unbalanced condition of feeder load and simultaneously reduce the power losses of ADNs. Then, the original non-convex model is converted into a second-order cone programming (SOCP) model using convex relaxation. In order to tighten the SOCP relaxation and improve the computation efficiency, an enhanced SOCP-based approach is developed to solve the proposed model. Finally, case studies are performed on the modified IEEE 33-node system to verify the effectiveness and efficiency of the proposed method.« less

Object categorization by wild ranging birds-Winter feeder experiments.

PubMed

Nováková, Nela; Veselý, Petr; Fuchs, Roman

2017-10-01

The object categorization is only scarcely studied using untrained wild ranging animals and relevant stimuli. We tested the importance of the spatial position of features salient for categorization of a predator using wild ranging birds (titmice) visiting a winter feeder. As a relevant stimulus we used a dummy of a raptor, the European sparrowhawk (Accipiter nisus), placed at the feeding location. This dummy was designed to be dismantled into three parts and rearranged with the head in the correct position, in the middle or at the bottom of the dummy. When the birds had the option of visiting an alternative feeder with a dummy pigeon, they preferred this option to visiting the feeder with the dummy sparrowhawk with the head in any of the three positions. When the birds had the option of visiting an alternative feeder with an un-rearranged dummy sparrowhawk, they visited both feeders equally often, and very scarcely. This suggests that the titmice considered all of the sparrowhawk modifications as being dangerous, and equally dangerous as the un-rearranged sparrowhawk. The position of the head was not the most important cue for categorization. The presence of the key features was probably sufficient for categorization, and their mutual spatial position was of lower importance. Copyright © 2017 Elsevier B.V. All rights reserved.

An enhanced SOCP-based method for feeder load balancing using the multi-terminal soft open point in active distribution networks

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

Ji, Haoran; Wang, Chengshan; Li, Peng

The integration of distributed generators (DGs) exacerbates the feeder power flow fluctuation and load unbalanced condition in active distribution networks (ADNs). The unbalanced feeder load causes inefficient use of network assets and network congestion during system operation. The flexible interconnection based on the multi-terminal soft open point (SOP) significantly benefits the operation of ADNs. The multi-terminal SOP, which is a controllable power electronic device installed to replace the normally open point, provides accurate active and reactive power flow control to enable the flexible connection of feeders. An enhanced SOCP-based method for feeder load balancing using the multi-terminal SOP is proposedmore » in this paper. Furthermore, by regulating the operation of the multi-terminal SOP, the proposed method can mitigate the unbalanced condition of feeder load and simultaneously reduce the power losses of ADNs. Then, the original non-convex model is converted into a second-order cone programming (SOCP) model using convex relaxation. In order to tighten the SOCP relaxation and improve the computation efficiency, an enhanced SOCP-based approach is developed to solve the proposed model. Finally, case studies are performed on the modified IEEE 33-node system to verify the effectiveness and efficiency of the proposed method.« less

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

PubMed

Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

2017-11-08

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

PubMed

Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques.

PubMed

Wales, Andrew; Breslin, Mark; Davies, Robert

2006-09-10

Salmonella infection of laying flocks in the UK is predominantly a problem of the persistent contamination of layer houses and associated wildlife vectors by Salmonella Enteritidis. Methods for its control and elimination include effective cleaning and disinfection of layer houses between flocks, and it is important to be able to measure the success of such decontamination. A method for the environmental detection and semi-quantitative enumeration of salmonellae was used and compared with a standard qualitative method, in 12 Salmonella-contaminated caged layer houses before and after cleaning and disinfection. The quantitative technique proved to have comparable sensitivity to the standard method, and additionally provided insights into the numerical Salmonella challenge that replacement flocks would encounter. Elimination of S. Enteritidis was not achieved in any of the premises examined although substantial reductions in the prevalence and numbers of salmonellae were demonstrated, whilst in others an increase in contamination was observed after cleaning and disinfection. Particular problems with feeders and wildlife vectors were highlighted. The use of a quantitative method assisted the identification of problem areas, such as those with a high initial bacterial load or those experiencing only a modest reduction in bacterial count following decontamination.

Effects of diet form and feeder adjustment on growth performance of nursery and finishing pigs.

PubMed

Nemechek, J E; Tokach, M D; Dritz, S S; Fruge, E D; Hansen, E L; Goodband, R D; DeRouchey, J M; Woodworth, J C

2015-08-01

Three experiments were conducted to determine the effects of feeder adjustment and diet form on growth performance of nursery (Exp. 1 and 2) and finishing (Exp. 3) pigs. Treatments were arranged as a 2 × 3 factorial with the main effects of feeder adjustment and diet form. The 2 feeder adjustments were a narrow and wide feeder adjustment (minimum gap opening of 1.27 and 2.54 cm, respectively). The 3 diet forms were meal, poor-quality pellets (70% pellets and 30% fines for Exp. 1 and 2 and 50% pellets and 50% fines for Exp. 3), and screened pellets with minimal fines (3 to 10%). In Exp. 1, 210 pigs (initially 11.9 kg BW) were used in a 21-d trial with 7 pigs per pen and 5 pens per treatment. No feeder adjustment × diet form interactions were observed. There were no differences in ADG, ADFI, or G:F due to feeder adjustment. Pigs fed the meal diet had increased ( < 0.05) ADG and ADFI compared with pigs fed the poor-quality or screened pellets. Pigs fed meal or poor-quality pellets had decreased ( < 0.05) G:F compared with pigs fed screened pellets. In Exp. 2, 1,005 nursery pigs (initially 14.1 kg BW) were used in a 28-d trial with 26 to 28 pigs per pen and 6 pens per treatment. Pigs fed from the narrow feeder adjustment had decreased ( < 0.05) ADG and ADFI compared with pigs fed from the wide adjustment with no differences in G:F. Pigs fed the meal diet had decreased ( < 0.05) ADG compared with pigs fed poor-quality or screened pellets. Pigs fed meal or poor-quality pellets had decreased ( < 0.05) G:F compared with pigs fed screened pellets. In Exp. 3, 246 pigs (initially 56.8 kg BW) were used in a 69-d trial with 5 pens per treatment and 6 or 7 pigs per pen. Overall, ADFI decreased ( < 0.05) and G:F increased ( < 0.05) for pigs fed from the narrow adjusted feeders compared with the wide adjustment with no differences in ADG. Overall, pigs fed meal diets tended to have decreased ( < 0.10) ADG and had decreased ( < 0.05) G:F compared with pigs fed screened pellets; ADG and G:F in those fed poor-quality pellets were intermediate. Feeding meal or poor-quality pellets increased ( < 0.05) ADFI compared with pigs fed screened pellets. In conclusion, feeding nursery pigs from a wide feeder gap may increase ADG and ADFI with no negative effects on G:F. For finishing pigs, reducing feeder gap reduced feed disappearance and improved G:F. In all experiments, the greatest G:F improvements from pelleting were observed when the percentage of fines was minimized.

Modern Grid Initiative Distribution Taxonomy Final Report

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

Schneider, Kevin P.; Chen, Yousu; Chassin, David P.

2008-11-01

This is the final report for the development of a toxonomy of prototypical electrical distribution feeders. Two of the primary goals of the Department of Energy's (DOE) Modern Grid Initiative (MGI) are 'to accelerate the modernization of our nation's electricity grid' and to 'support demonstrations of systems of key technologies that can serve as the foundation for an integrated, modern power grid'. A key component to the realization of these goals is the effective implementation of new, as well as existing, 'smart grid technologies'. Possibly the largest barrier that has been identified in the deployment of smart grid technologies ismore » the inability to evaluate how their deployment will affect the electricity infrastructure, both locally and on a regional scale. The inability to evaluate the impacts of these technologies is primarily due to the lack of detailed electrical distribution feeder information. While detailed distribution feeder information does reside with the various distribution utilities, there is no central repository of information that can be openly accessed. The role of Pacific Northwest National Laboratory (PNNL) in the MGI for FY08 was to collect distribution feeder models, in the SynerGEE{reg_sign} format, from electric utilities around the nation so that they could be analyzed to identify regional differences in feeder design and operation. Based on this analysis PNNL developed a taxonomy of 24 prototypical feeder models in the GridLAB-D simulations environment that contain the fundamental characteristics of non-urban core, radial distribution feeders from the various regions of the U.S. Weighting factors for these feeders are also presented so that they can be used to generate a representative sample for various regions within the United States. The final product presented in this report is a toolset that enables the evaluation of new smart grid technologies, with the ability to aggregate their effects to regional and national levels. The distribution feeder models presented in this report are based on actual utility models but do not contain any proprietary or system specific information. As a result, the models discussed in this report can be openly distributed to industry, academia, or any interested entity, in order to facilitate the ability to evaluate smart grid technologies.« less

Development of an ES-like cell culture system (RESC) from rohu, Labeo rohita (Ham.).

PubMed

Goswami, M; Lakra, W S; Yadav, Kamalendra; Jena, J K

2012-12-01

An embryonic stem (ES)-like cell culture system RESC from a commercially important freshwater carp, Labeo rohita, was developed using blastula stage embryos. The cells were cultured in Leibovitz-15 (L-15) medium in gelatin-coated cell culture flask supplemented with 15 % fetal bovine serum along with 10 ng ml(-1) basic fibroblast growth factor at 28 °C under feeder-free conditions. The ES-like cells were characterized by their unique morphology, alkaline phosphatase activity, embryoid body formation tendency, expression of transcription factor Oct4, and consistent chromosome count. The RESC cells when treated with retinoic acid differentiated into cells of different lineages. The RESC developed from mid-blastula embryos of L. rohita would be a useful tool for cellular differentiation and gene expression studies.

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

PubMed Central

Takenaka, Chiemi; Miyajima, Hiroshi; Yoda, Yusuke; Imazato, Hideo; Yamamoto, Takako; Gomi, Shinichi; Ohshima, Yasuhiro; Kagawa, Kenichi; Sasaki, Tetsuji; Kawamata, Shin

2015-01-01

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control. PMID:26115194

Mesenchymal stem cells from the Wharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture

PubMed Central

Bakhshi, Tiki; Zabriskie, Ryan C.; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A.; Gregory, Stephanie A.; Fung, Henry C.; Christopherson, Kent W.

2012-01-01

BACKGROUND Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow–derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton’s jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. STUDY DESIGN AND METHODS Umbilical cord–derived MSCs (UC-MSCs) were cultured from the Wharton’s jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. RESULTS Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. CONCLUSION These data suggest the potential therapeutic application of Wharton’s jelly–derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation. PMID:18798803

Can rats solve a simple version of the traveling salesman problem?

PubMed

Bures, J; Buresová, O; Nerad, L

1992-12-31

Whereas correct tours through the radial arm maze are almost equally long, free choice mazes with multiple goals scattered in an open field allow the animal to select the shortest one from a multitude of correct tours. Thirteen rats were trained (at 10 trials per day) to visit an array of cylindrical feeders in an open field (40 x 100 cm) with reward available only when visiting the last feeder of the set. In Expt. 1 with eight feeders arranged in five different configurations the rats made after 10 days of training 1 error in the first 8 choices and incidence of errorless trials was about 20%. In Expt. 2. the use of six feeders in a rectangular (A) or double triangle (B) configuration increased the incidence of errorless trials to 60%. Expt. 3 showed that performance in the 6-feeder maze was significantly impaired by 6 mg/kg ketamine or 0.25 mg/kg scopolamine but not by lower dosages of these drugs. Tours generated on errorless trials (each feeder visited only once) during 10 days of Expt. 2 were analyzed. Six places can be visited in 6! = 720 different closed tours the lengths of which (in arbitrary units) range from 6.00 to 10.12 for A and from 6.83 to 10.47 for B. Whereas random generation of correct routes yielded only 5% of the shortest tours, they were clearly preferred by rats (41% in A and 45% in B). The apparent proficiency of rats in this optimization problem is probably not due to cognitive comparison of the possible correct routes but rather to following a simple rule 'Always go to the nearest not yet visited feeder'.

47 CFR 25.250 - Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 2 2014-10-01 2014-10-01 false Sharing between NGSO MSS Feeder links Earth....250 Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands. (a) NGSO MSS applicants shall be licensed to operate in the 29.1-29.5 GHz band for Earth-to-space...

47 CFR 25.250 - Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 2 2013-10-01 2013-10-01 false Sharing between NGSO MSS Feeder links Earth....250 Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands. (a) NGSO MSS applicants shall be licensed to operate in the 29.1-29.5 GHz band for Earth-to-space...

47 CFR 25.250 - Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 2 2012-10-01 2012-10-01 false Sharing between NGSO MSS Feeder links Earth....250 Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands. (a) NGSO MSS applicants shall be licensed to operate in the 29.1-29.5 GHz band for Earth-to-space...

47 CFR 25.250 - Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 2 2010-10-01 2010-10-01 false Sharing between NGSO MSS Feeder links Earth....250 Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands. (a) NGSO MSS applicants shall be licensed to operate in the 29.1-29.5 GHz band for Earth-to-space...

47 CFR 25.250 - Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 2 2011-10-01 2011-10-01 false Sharing between NGSO MSS Feeder links Earth....250 Sharing between NGSO MSS Feeder links Earth Stations in the 19.3-19.7 GHz and 29.1-29.5 GHz Bands. (a) NGSO MSS applicants shall be licensed to operate in the 29.1-29.5 GHz band for Earth-to-space...

Conceptual Design and Analysis of Cold Mass Support of the CS3U Feeder for the ITER

NASA Astrophysics Data System (ADS)

Zhu, Yinfeng; Song, Yuntao; Zhang, Yuanbin; Wang, Zhongwei

2013-06-01

In the International Thermonuclear Experimental Reactor (ITER) project, the feeders are one of the most important and critical systems. To convey the power supply and the coolant for the central solenoid (CS) magnet, 6 sets of CS feeders are employed, which consist mainly of an in-cryostat feeder (ICF), a cryostat feed-through (CFT), an S-bend box (SBB), and a coil terminal box (CTB). To compensate the displacements of the internal components of the CS feeders during operation, sliding cold mass supports consisting of a sled plate, a cylindrical support, a thermal shield, and an external ring are developed. To check the strength of the developed cold mass supports of the CS3U feeder, electromagnetic analysis of the two superconducting busbars is performed by using the CATIA V5 and ANSYS codes based on parametric technology. Furthermore, the thermal-structural coupling analysis is performed based on the obtained results, except for the stress concentration, and the max. stress intensity is lower than the allowable stress of the selected material. It is found that the conceptual design of the cold mass support can satisfy the required functions under the worst case of normal working conditions. All these performed activities will provide a firm technical basis for the engineering design and development of cold mass supports.

30 CFR 75.1001 - Overcurrent protection.

Code of Federal Regulations, 2011 CFR

2011-07-01

... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires § 75.1001 Overcurrent protection. [Statutory Provisions] Trolley wires and trolley feeder wires shall be provided with...

30 CFR 75.1001 - Overcurrent protection.

Code of Federal Regulations, 2010 CFR

2010-07-01

... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires § 75.1001 Overcurrent protection. [Statutory Provisions] Trolley wires and trolley feeder wires shall be provided with...

Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

PubMed

Pick, Marjorie

2016-01-01

Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use.

The influence of laser alloying on the structure and mechanical properties of AlMg5Si2Mn surface layers

NASA Astrophysics Data System (ADS)

Pakieła, W.; Tański, T.; Brytan, Z.; Labisz, K.

2016-04-01

The goal of this paper was focused on investigation of microstructure and properties of surface layer produced during laser surface treatment of aluminium alloy by high-power fibre laser. The performed laser treatment involves remelting and feeding of Inconel 625 powder into the aluminium surface. As a base metal was used aluminium alloy AlMg5Si2Mn. The Inconel powder was injected into the melt pool and delivered by a vacuum feeder at a constant rate of 4.5 g/min. The size of Inconel alloying powder was in the range 60-130 µm. In order to remelt the aluminium alloy surface, the fibre laser of 3 kW laser beam power has been used. The linear laser scan rate of the beam was set 0.5 m/min. Based on performed investigations, it was possible to obtain the layer consisting of heat-affected zone, transition zone and remelted zone, without cracks and defects having much higher hardness value compared to the non-alloyed material.

30 CFR 75.1000 - Cutout switches.

Code of Federal Regulations, 2011 CFR

2011-07-01

... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires § 75.1000 Cutout switches. [Statutory Provisions] Trolley wires and trolley feeder wires, shall be provided with cutout...

30 CFR 75.1000 - Cutout switches.

Code of Federal Regulations, 2010 CFR

2010-07-01

... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires § 75.1000 Cutout switches. [Statutory Provisions] Trolley wires and trolley feeder wires, shall be provided with cutout...

Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

PubMed Central

Yanagi, Satoshi; Kato, Chika; Takashima, Ryokichi; Kobayashi, Eiji; Hagiwara, Keitaro; Ochiya, Takahiro

2015-01-01

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation. PMID:25875613

Nematode communities in sediments of the Kermadec Trench, Southwest Pacific Ocean

NASA Astrophysics Data System (ADS)

Leduc, Daniel; Rowden, Ashley A.

2018-04-01

Hadal trenches are characterized by environmental conditions not found in any other deep-sea environment, such as steep topography and periodic disturbance by turbidity flows, which are likely responsible for the distinct nature of benthic communities of hadal trenches relative to those of the abyssal plain. Nematodes are the most abundant metazoans in the deep-sea benthos, but it is not yet clear if different trenches host distinct nematode communities, and no data are yet available on the communities of most trenches, including the Kermadec Trench in the Southwest Pacific. Quantitative core samples from the seafloor of the Kermadec Trench were recently obtained from four sites at 6000-9000 m depth which allowed for analyses of meiofauna, and nematodes in particular, for the first time. Nematode community and trophic structure was also compared with other trenches using published data. There was a bathymetric gradient in meiofauna abundance, biomass, and community structure within the Kermadec Trench, but patterns for species richness were ambiguous depending on which metric was used. There was a change in community structure from shallow to deep sites, as well as a consistent change in community structure from the upper sediment layers to the deeper sediment layers across the four sites. These patterns are most likely explained by variation in food availability within the trench, and related to trench topography. Together, deposit and microbial feeders represented 48-92% of total nematode abundance in the samples, which suggests that fine organic detritus and bacteria are major food sources. The relatively high abundance of epigrowth feeders at the 6000 and 9000 m sites (38% and 31%, respectively) indicates that relatively freshly settled microalgal cells represent another important food source at these sites. We found a significant difference in species community structure between the Kermadec and Tonga trenches, which was due to both the presence/absence of species as well as differences in relative abundances of shared species. The cluster and SIMPROF analyses of nematode genus community data across Pacific and Atlantic trenches identified two statistically significant natural groupings: the first group comprised all three Puerto Rico Trench samples, and the second comprised all remaining trenches (South Sandwich, Atacama, Tonga, and Kermadec). Our analyses show that differences in nematode between the adjacent Kermadec and Tonga trenches are observable when analyses are conducted with species-level identifications, but genera-based and trophic structure analyses revealed only limited heterogeneity among trenches. The present study contributes to the growing amount of information on hadal trench environments, which ultimately will build a greater understanding of these rarely sampled deep-sea habitats.

Distribution Feeder Modeling for Time-Series Simulation of Voltage Management Strategies: Preprint

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

Giraldez Miner, Julieta I; Gotseff, Peter; Nagarajan, Adarsh

This paper presents techniques to create baseline distribution models using a utility feeder from Hawai'ian Electric Company. It describes the software-to-software conversion, steady-state, and time-series validations of a utility feeder model. It also presents a methodology to add secondary low-voltage circuit models to accurately capture the voltage at the customer meter level. This enables preparing models to perform studies that simulate how customer-sited resources integrate into legacy utility distribution system operations.

Breast implant-associated, ALK-negative, T-cell, anaplastic, large-cell lymphoma: establishment and characterization of a model cell line (TLBR-1) for this newly emerging clinical entity.

PubMed

Lechner, Melissa G; Lade, Stephen; Liebertz, Daniel J; Prince, H Miles; Brody, Garry S; Webster, Howard R; Epstein, Alan L

2011-04-01

Primary lymphomas of the breast are very rare (0.2-1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)(+) and perforin(+) . Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR(+) CD80(+) CD86(+) ), IL-2 signaling (CD25(+) CD122(+) ), and NK (CD56(+) ) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized disease entity. Copyright © 2010 American Cancer Society.

Breast Implant-Associated, ALK-Negative, T-Cell, Anaplastic, Large-Cell Lymphoma: Establishment and Characterization of a Model Cell Line (TLBR-1) for This Newly Emerging Clinical Entity

PubMed Central

Lechner, Melissa G.; Lade, Stephen; Liebertz, Daniel J.; Prince, H. Miles; Brody, Garry S.; Webster, Howard R.; Epstein, Alan L.

2014-01-01

BACKGROUND Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. METHODS A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. RESULTS Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)+ and perforin+. Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR+CD80+CD86+), IL-2 signaling (CD25+CD122+), and NK (CD56+) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. CONCLUSIONS TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized disease entity. PMID:21425149

21 CFR 520.1448a - Monensin blocks.

Code of Federal Regulations, 2011 CFR

2011-04-01

...) Limitations. Block to be fed free choice to pasture cattle (slaughter, stocker, feeder, and dairy and beef.... Blocks to be fed free choice to pasture cattle (slaughter, stocker, feeder, and dairy and beef...

11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND ...

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

11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND BUILT BY WES. - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

Impact of Distribution Feeders that do not have Voltage Regulators on the number of Charged Electric Vehicles using IEEE 34 Bus Test Feeder

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

Allehyani, Ahmed; Beshir, Mohammed

Voltage regulators help maintain an acceptable voltage profile for the system. This paper discusses the effect of installing voltage regulators to the system to fix the voltage drop resulting from the electrical vehicles loading increase when they are being charged. The effect will be studied in the afternoon, when the peak load occurs, using the IEEE 34 bus test feeder. First, only one spot node is used to charge the electric vehicles while a voltage regulator is present. Second, five spot nodes are loaded at the same time to charge the electric vehicles while voltage regulators are installed at eachmore » node. After that, the impact of electric vehicles on distribution feeders that do not have voltage regulators will appear.« less

IEEE 342 Node Low Voltage Networked Test System

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

Schneider, Kevin P.; Phanivong, Phillippe K.; Lacroix, Jean-Sebastian

The IEEE Distribution Test Feeders provide a benchmark for new algorithms to the distribution analyses community. The low voltage network test feeder represents a moderate size urban system that is unbalanced and highly networked. This is the first distribution test feeder developed by the IEEE that contains unbalanced networked components. The 342 node Low Voltage Networked Test System includes many elements that may be found in a networked system: multiple 13.2kV primary feeders, network protectors, a 120/208V grid network, and multiple 277/480V spot networks. This paper presents a brief review of the history of low voltage networks and how theymore » evolved into the modern systems. This paper will then present a description of the 342 Node IEEE Low Voltage Network Test System and power flow results.« less

Comparing the engineering program feeders from SiF and convention models

NASA Astrophysics Data System (ADS)

Roongruangsri, Warawaran; Moonpa, Niwat; Vuthijumnonk, Janyawat; Sangsuwan, Kampanart

2018-01-01

This research aims to compare the relationship between two types of engineering program feeder models within the technical education systems of Rajamangala University of Technology Lanna (RMUTL), Chiangmai, Thailand. To illustrate, the paper refers to two typologies of feeder models, which are the convention and the school in factory (SiF) models. The new SiF model is developed through a collaborative educational process between the sectors of industry, government and academia, using work-integrated learning. The research methodology were use to compared features of the the SiF model with conventional models in terms of learning outcome, funding budget for the study, the advantages and disadvantages from the point of view of students, professors, the university, government and industrial partners. The results of this research indicate that the developed SiF feeder model is the most pertinent ones as it meet the requirements of the university, the government and the industry. The SiF feeder model showed the ability to yield positive learning outcomes with low expenditures per student for both the family and the university. In parallel, the sharing of knowledge between university and industry became increasingly important in the process, which resulted in the improvement of industrial skills for professors and an increase in industrial based research for the university. The SiF feeder model meets its demand of public policy in supporting a skilled workforce for the industry, which could be an effective tool for the triple helix educational model of Thailand.

Complex memories in honeybees: can there be more than two?

PubMed

Reinhard, Judith; Srinivasan, Mandyam V; Zhang, Shaowu

2006-04-01

Foraging honeybees are likely to learn visual and chemical cues associated with many different food sources. Here, we explore how many such sources can be memorized and recalled. Marked bees were trained to visit two (or three) sugar feeders, each placed at a different outdoor location and carrying a different scent. We then tested the ability of the bees to recall these locations and fly to them, when the training scents were blown into the hive, and the scents and food at the feeders were removed. When trained on two feeder locations, each associated with a different scent, the bees could correctly recall the location associated with each scent. However, this ability broke down when the number of scents and feeder locations was increased to three. Performance was partially restored when each of the three training feeders was endowed with an additional cue, namely, a distinct colour. Our results suggest that bees can recall a maximum of two locations when each is associated with a different scent. However, this number can be increased if the scent cues are augmented by visual cues. These findings have implications for the ways in which associations are established and laid down in honeybee memory.

Optic flow informs distance but not profitability for honeybees.

PubMed

Shafir, Sharoni; Barron, Andrew B

2010-04-22

How do flying insects monitor foraging efficiency? Honeybees (Apis mellifera) use optic flow information as an odometer to estimate distance travelled, but here we tested whether optic flow informs estimation of foraging costs also. Bees were trained to feeders in flight tunnels such that bees experienced the greatest optic flow en route to the feeder closest to the hive. Analyses of dance communication showed that, as expected, bees indicated the close feeder as being further, but they also indicated this feeder as the more profitable, and preferentially visited this feeder when given a choice. We show that honeybee estimates of foraging cost are not reliant on optic flow information. Rather, bees can assess distance and profitability independently and signal these aspects as separate elements of their dances. The optic flow signal is sensitive to the nature of the environment travelled by the bee, and is therefore not a good index of flight energetic costs, but it provides a good indication of distance travelled for purpose of navigation and communication, as long as the dancer and recruit travel similar routes. This study suggests an adaptive dual processing system in honeybees for communicating and navigating distance flown and for evaluating its energetic costs.

Optic flow informs distance but not profitability for honeybees

PubMed Central

Shafir, Sharoni; Barron, Andrew B.

2010-01-01

How do flying insects monitor foraging efficiency? Honeybees (Apis mellifera) use optic flow information as an odometer to estimate distance travelled, but here we tested whether optic flow informs estimation of foraging costs also. Bees were trained to feeders in flight tunnels such that bees experienced the greatest optic flow en route to the feeder closest to the hive. Analyses of dance communication showed that, as expected, bees indicated the close feeder as being further, but they also indicated this feeder as the more profitable, and preferentially visited this feeder when given a choice. We show that honeybee estimates of foraging cost are not reliant on optic flow information. Rather, bees can assess distance and profitability independently and signal these aspects as separate elements of their dances. The optic flow signal is sensitive to the nature of the environment travelled by the bee, and is therefore not a good index of flight energetic costs, but it provides a good indication of distance travelled for purpose of navigation and communication, as long as the dancer and recruit travel similar routes. This study suggests an adaptive dual processing system in honeybees for communicating and navigating distance flown and for evaluating its energetic costs. PMID:20018787

Energy efficiency analysis of two-sided feed scheme of DC traction network with high asymmetry of feeders parameters

NASA Astrophysics Data System (ADS)

Abramov, E. Y.; Sopov, V. I.

2017-10-01

In a given research using the example of traction network area with high asymmetry of power supply parameters, the sequence of comparative assessment of power losses in DC traction network with parallel and traditional separated operating modes of traction substation feeders was shown. Experimental measurements were carried out under these modes of operation. The calculation data results based on statistic processing showed the power losses decrease in contact network and the increase in feeders. The changes proved to be critical ones and this demonstrates the significance of potential effects when converting traction network areas into parallel feeder operation. An analytical method of calculation the average power losses for different feed schemes of the traction network was developed. On its basis, the dependences of the relative losses were obtained by varying the difference in feeder voltages. The calculation results showed unreasonableness transition to a two-sided feed scheme for the considered traction network area. A larger reduction in the total power loss can be obtained with a smaller difference of the feeders’ resistance and / or a more symmetrical sectioning scheme of contact network.

Urban and rural habitats differ in number and type of bird feeders and in bird species consuming supplementary food.

PubMed

Tryjanowski, Piotr; Skórka, Piotr; Sparks, Tim H; Biaduń, Waldemar; Brauze, Tomasz; Hetmański, Tomasz; Martyka, Rafał; Indykiewicz, Piotr; Myczko, Łukasz; Kunysz, Przemysław; Kawa, Piotr; Czyż, Stanisław; Czechowski, Paweł; Polakowski, Michał; Zduniak, Piotr; Jerzak, Leszek; Janiszewski, Tomasz; Goławski, Artur; Duduś, Leszek; Nowakowski, Jacek J; Wuczyński, Andrzej; Wysocki, Dariusz

2015-10-01

Bird feeding is one of the most widespread direct interactions between man and nature, and this has important social and environmental consequences. However, this activity can differ between rural and urban habitats, due to inter alia habitat structure, human behaviour and the composition of wintering bird communities. We counted birds in 156 squares (0.25 km(2) each) in December 2012 and again in January 2013 in locations in and around 26 towns and cities across Poland (in each urban area, we surveyed 3 squares and also 3 squares in nearby rural areas). At each count, we noted the number of bird feeders, the number of bird feeders with food, the type of feeders, additional food supplies potentially available for birds (bread offered by people, bins) and finally the birds themselves. In winter, urban and rural areas differ in the availability of food offered intentionally and unintentionally to birds by humans. Both types of food availability are higher in urban areas. Our findings suggest that different types of bird feeder support only those species specialized for that particular food type and this relationship is similar in urban and rural areas.

View northwest, discharge basin, floor, showing cement cross beams built ...

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

View northwest, discharge basin, floor, showing cement cross beams built on stone bases - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

The effect of different biologic and biosynthetic wound covers on keratinocyte growth, stratification and differentiation in vitro

PubMed Central

Mestak, Ondrej

2014-01-01

The purpose of this study was to compare, by means of in vitro cultivation technique, five marketed brands of wound covers used in the treatment of burns and other skin defects (Biobrane®, Suprathel®, Veloderm®, Xe-Derma®, and Xenoderm®) for their ability to stimulate the keratinocyte growth, stratification, and differentiation. In three independent experiments, human keratinocytes were grown on the tested covers in organotypic cultures by the 3T3 feeder layer technique. Vertical paraffin sections of the wound covers with keratinocytes were processed using hematoxylin–eosin staining and immunostaining for involucrin. Keratinocyte populations on the dressings were assessed for (1) number of keratinocyte strata (primary variable), (2) quantitative growth, (3) thickness of the keratinocyte layer, and (4) cell differentiation. The Xe-Derma wound cover provided the best support to keratinocyte proliferation and stratification, with the number of keratinocyte strata significantly (p < 0.05) higher in comparison to all products studied, except Xenoderm. However, in contrast to Xe-Derma, Xenoderm did not significantly differ from the other dressings. The results of this in vitro study show that the brands based on porcine dermal matrix possess the strongest effect on keratinocyte proliferation and stratification. The distinctive position of Xe-Derma may be related to its composition, where natural dermal fibers form a smooth surface, similar to the basement membrane. Furthermore, the results indicate that in vitro evaluation of effects on epithelial growth may accelerate the development of new bio-engineering-based wound covers. PMID:25383177

Spacelab 4: Primate experiment support hardware

NASA Astrophysics Data System (ADS)

Fusco, P. R.; Peyran, R. J.

1984-05-01

A squirrel monkey feeder and automatic urine collection system were designed to fly on the Spacelab 4 Shuttle Mission presently scheduled for January 1986. Prototypes of the feeder and urine collection systems were fabricated and extensively tested on squirrel monkeys at the National Aeronautics and Space Administration's (NASA) Ames Research Center (ARC). The feeder design minimizes impact on the monkey's limited space in the cage and features improved reliability and biocompatibility over previous systems. The urine collection system is the first flight qualified, automatic urine collection device for squirrel monkeys. Flight systems are currently being fabricated.

Spacelab 4: Primate experiment support hardware

NASA Technical Reports Server (NTRS)

Fusco, P. R.; Peyran, R. J.

1984-01-01

A squirrel monkey feeder and automatic urine collection system were designed to fly on the Spacelab 4 Shuttle Mission presently scheduled for January 1986. Prototypes of the feeder and urine collection systems were fabricated and extensively tested on squirrel monkeys at the National Aeronautics and Space Administration's (NASA) Ames Research Center (ARC). The feeder design minimizes impact on the monkey's limited space in the cage and features improved reliability and biocompatibility over previous systems. The urine collection system is the first flight qualified, automatic urine collection device for squirrel monkeys. Flight systems are currently being fabricated.

Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

PubMed

Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

2015-01-01

The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.

View southeast, interior of second drop, foundation and footwall and ...

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

View southeast, interior of second drop, foundation and footwall and sidewalls, showing failed lower arch - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

Effects of soil compaction on root and root hair morphology: implications for campsite rehabilitation

Treesearch

L. Alessa; C. G. Earnhart

2000-01-01

Recreational use of wild lands can create areas, such as campsites, which may experience soil compaction and a decrease in vegetation cover and diversity. Plants are highly reliant on their roots’ ability to uptake nutrients and water from soil. Any factors that affect the highly specialized root hairs (“feeder cells”) compromise the overall health and survival of the...

Extremum Seeking Control of Smart Inverters for VAR Compensation

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

Arnold, Daniel; Negrete-Pincetic, Matias; Stewart, Emma

2015-09-04

Reactive power compensation is used by utilities to ensure customer voltages are within pre-defined tolerances and reduce system resistive losses. While much attention has been paid to model-based control algorithms for reactive power support and Volt Var Optimization (VVO), these strategies typically require relatively large communications capabilities and accurate models. In this work, a non-model-based control strategy for smart inverters is considered for VAR compensation. An Extremum Seeking control algorithm is applied to modulate the reactive power output of inverters based on real power information from the feeder substation, without an explicit feeder model. Simulation results using utility demand informationmore » confirm the ability of the control algorithm to inject VARs to minimize feeder head real power consumption. In addition, we show that the algorithm is capable of improving feeder voltage profiles and reducing reactive power supplied by the distribution substation.« less

Mass extinctions: Ecological selectivity and primary production

NASA Astrophysics Data System (ADS)

Rhodes, Melissa Clark; Thayer, Charles W.

1991-09-01

If mass extinctions were caused by reduced primary productivity, then extinctions should be concentrated among animals with starvation-susceptible feeding modes, active lifestyles, and high-energy budgets. The stratigraphic ranges (by stage) of 424 genera of bivalves and 309 genera of articulate brachiopods suggest that there was an unusual reduction of primary productivity at the Cretaceous/Tertiary (K/T) boundary extinction. For bivalves at the K/T, there were (1) selective extinction of suspension feeders and other susceptible trophic categories relative to deposit feeders and other resistant categories, and (2) among suspension feed-ers, selective extinction of bivalves with active locomotion. During the Permian-Triassic (P/Tr) extinction and Jurassic background time, extinction rates among suspension feeders were greater for articulate brachiopods than for bivalves. But during the K/T event, extinction rates of articulates and suspension-feeding bivalves equalized, possibly because the low-energy budgets of articulates gave them an advantage when food was scarce.

Subjective value of risky foods for individual domestic chicks: a hierarchical Bayesian model.

PubMed

Kawamori, Ai; Matsushima, Toshiya

2010-05-01

For animals to decide which prey to attack, the gain and delay of the food item must be integrated in a value function. However, the subjective value is not obtained by expected profitability when it is accompanied by risk. To estimate the subjective value, we examined choices in a cross-shaped maze with two colored feeders in domestic chicks. When tested by a reversal in food amount or delay, chicks changed choices similarly in both conditions (experiment 1). We therefore examined risk sensitivity for amount and delay (experiment 2) by supplying one feeder with food of fixed profitability and the alternative feeder with high- or low-profitability food at equal probability. Profitability varied in amount (groups 1 and 2 at high and low variance) or in delay (group 3). To find the equilibrium, the amount (groups 1 and 2) or delay (group 3) of the food in the fixed feeder was adjusted in a total of 18 blocks. The Markov chain Monte Carlo method was applied to a hierarchical Bayesian model to estimate the subjective value. Chicks undervalued the variable feeder in group 1 and were indifferent in group 2 but overvalued the variable feeder in group 3 at a population level. Re-examination without the titration procedure (experiment 3) suggested that the subjective value was not absolute for each option. When the delay was varied, the variable option was often given a paradoxically high value depending on fixed alternative. Therefore, the basic assumption of the uniquely determined value function might be questioned.

Feeder systems of acidic lava flows from the Paraná-Etendeka Igneous Province in southern Brazil and their implications for eruption style

NASA Astrophysics Data System (ADS)

de Lima, Evandro Fernandes; Waichel, Breno Leitão; Rossetti, Lucas De Magalhães May; Sommer, Carlos Augusto; Simões, Matheus Silva

2018-01-01

In the Rio Grande do Sul State, southern Brazil, the volcanic sequence of the Paraná-Etendeka Igneous Province consists of pahoehoe and rubbly pahoehoe lava flows with basaltic and basaltic andesitic composition respectively, overlaid by acidic volcanic rocks. The acidic volcanic rocks of the Paraná-Etendeka Igneous Province exhibit textures and structures that can be related to effusive and/or explosive eruptions generating predominantly rheoignimbrites. The huge lava volume related to the emplacement of large igneous provinces implicates on efficient feeder systems that are more commonly observed in continental environments. In the Paraná-Etendeka Igneous Province, feeders of basaltic rocks are exposed in several dyke swarms (Ponta Grossa NW trending, Florianópolis/Skeleton Coast (NW Namibia) N-S trending, Serra do Mar NE trending and Henties Bay/Outjo NE trending). In contrast, the only feeder system proposed to the acidic rocks of the Paraná-Etendeka Igneous Province is the Messum complex in Namibia (Milner et al. 1995). In the study area, the opening of three quarries for the extraction of dimension stones has exposed impressive structures/textures that show the effusive emplacement and the ductile to fragile-ductile magma transition along the acidic feeder dykes. Besides that, magma mixing/mingling processes between two acidic magmas are observed along the dykes. Here we describe new occurrences of acidic feeder dykes, correlate the dykes with acidic flows and discuss their importance to understand the emplacement of the Palmas type acid units in southern Brazil.

2. Photocopy of a drawing (original in the Collection of ...

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

2. Photocopy of a drawing (original in the Collection of the PL&C, Shelf 117, Drawing 155) SECTION OF THE MOODY STREET FEEDER, DECEMBER 30, 1847 - Moody Street Feeder, Moody Street vicinity, Lowell, Middlesex County, MA

30 CFR 77.1801 - Overcurrent protection.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Overcurrent protection. 77.1801 Section 77.1801... Wires and Trolley Feeder Wires § 77.1801 Overcurrent protection. Trolley wires and trolley feeder wires shall be provided with overcurrent protection. ...

Treatment of Silk Fibroin with Poly(ethylene glycol) for the Enhancement of Corneal Epithelial Cell Growth

PubMed Central

Suzuki, Shuko; Dawson, Rebecca A.; Chirila, Traian V.; Shadforth, Audra M. A.; Hogerheyde, Thomas A.; Edwards, Grant A.; Harkin, Damien G.

2015-01-01

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes. PMID:26034883

Microstructure and properties of laser-clad high-temperature wear-resistant alloys

NASA Astrophysics Data System (ADS)

Yang, Yongqiang

1999-02-01

A 2-kW CO 2 laser with a powder feeder was used to produce alloy coatings with high temperature-wear resistance on the surface of steel substrates. To analyze the microstructure and microchemical composition of the laser-clad layers, a scanning electron microscope (SEM) equipped with an energy dispersive X-ray microanalysis system was employed. X-ray diffraction techniques were applied to characterize the phases formed during the cladding process. The results show that the microstructure of the cladding alloy consists mainly of many dispersed particles (W 2C, (W,Ti)C 1- x, WC), a lamellar eutectic carbide M 12C, and an (f.c.c) matrix. Hardness tested at room and high temperature showed that the laser-clad zone has a moderate room temperature hardness and relatively higher elevated temperature hardness. The application of the laser-clad layer to a hot tool was very successful, and its operational life span was prolonged 1 to 4 times.

47 CFR 25.147 - Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz.

Code of Federal Regulations, 2014 CFR

2014-10-01

... COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Applications and Licenses Space Stations § 25.147 Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz. If an NGSO...

47 CFR 25.147 - Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz.

Code of Federal Regulations, 2012 CFR

2012-10-01

... COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Applications and Licenses Space Stations § 25.147 Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz. If an NGSO...

47 CFR 25.147 - Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz.

Code of Federal Regulations, 2010 CFR

2010-10-01

... COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Applications and Licenses Space Stations § 25.147 Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz. If an NGSO...

47 CFR 25.147 - Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz.

Code of Federal Regulations, 2011 CFR

2011-10-01

... COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Applications and Licenses Space Stations § 25.147 Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz. If an NGSO...

47 CFR 25.147 - Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz.

Code of Federal Regulations, 2013 CFR

2013-10-01

... COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Applications and Licenses Space Stations § 25.147 Licensing provision for NGSO MSS feeder downlinks in the band 6700-6875 MHz. If an NGSO...

Evaluation of Advanced Microwave Landing System Procedures in the New York Terminal Area

DTIC Science & Technology

1991-03-01

sector controller called the CAMRN sector who must then sequence that traffic with multiple feeders from the south before handing off to the final...Right (13R) were all being used by landing traffic, the final controller handled the runway 22 arrivals and the CAMRN controller handled the runway 13R...Feeder Fix AAL678 DC10 H 00:09:00 AAL68 B767 H 00:23:00 AAL588 A300 H 00:27:00 PAA224 A300 H 01:20:00 4/ TWAll L101 H 01:34:00 CAMRN Feeder Fix DAL144

Vertical-Screw-Auger Conveyer Feeder

NASA Technical Reports Server (NTRS)

Walton, Otis (Inventor); Vollmer, Hubert J. (Inventor)

2016-01-01

A conical feeder is attached to a vertically conveying screw auger. The feeder is equipped with scoops and rotated from the surface to force-feed regolith the auger. Additional scoops are possible by adding a cylindrical section above the conical funnel section. Such then allows the unit to collect material from swaths larger in diameter than the enclosing casing pipe of the screw auger. A third element includes a flexible screw auger. All three can be used in combination in microgravity and zero atmosphere environments to drill and recover a wide area of subsurface regolith and entrained volatiles through a single access point on the surface.

Cost Benefit and Alternatives Analysis of Distribution Systems with Energy Storage Systems: Preprint

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

Harris, Tom; Nagarajan, Adarsh; Baggu, Murali

This paper explores monetized and non-monetized benefits from storage interconnected to distribution system through use cases illustrating potential applications for energy storage in California's electric utility system. This work supports SDG&E in its efforts to quantify, summarize, and compare the cost and benefit streams related to implementation and operation of energy storage on its distribution feeders. This effort develops the cost benefit and alternatives analysis platform, integrated with QSTS feeder simulation capability, and analyzed use cases to explore the cost-benefit of implementation and operation of energy storage for feeder support and market participation.

Microstrip Patch Antenna And Method

NASA Technical Reports Server (NTRS)

Fink, Patrick W. (Inventor)

2001-01-01

Method and apparatus are provided for a microstrip feeder structure for supplying properly phased signals to each radiator element in a microstrip antenna array that may be utilized for radiating circularly polarized electromagnetic waves. In one disclosed embodiment. the microstrip feeder structure includes a plurality of microstrip sections many or all of which preferably have an electrical length substantially equal to one-quarter wavelength at the antenna operating frequency. The feeder structure provides a low loss feed structure that may be duplicated multiple times through a set of rotations and translations to provide a radiating array of the desired size.

Movement of feeder-using songbirds: the influence of urban features.

PubMed

Cox, Daniel T C; Inger, Richard; Hancock, Steven; Anderson, Karen; Gaston, Kevin J

2016-11-23

Private gardens provide vital opportunities for people to interact with nature. The most popular form of interaction is through garden bird feeding. Understanding how landscape features and seasons determine patterns of movement of feeder-using songbirds is key to maximising the well-being benefits they provide. To determine these patterns we established three networks of automated data loggers along a gradient of greenspace fragmentation. Over a 12-month period we tracked 452 tagged blue tits Cyantistes caeruleus and great tits Parus major moving between feeder pairs 9,848 times, to address two questions: (i) Do urban features within different forms, and season, influence structural (presence-absence of connections between feeders by birds) and functional (frequency of these connections) connectivity? (ii) Are there general patterns of structural and functional connectivity across forms? Vegetation cover increased connectivity in all three networks, whereas the presence of road gaps negatively affected functional but not structural connectivity. Across networks structural connectivity was lowest in the summer when birds maintain breeding territories, however patterns of functional connectivity appeared to vary with habitat fragmentation. Using empirical data this study shows how key urban features and season influence movement of feeder-using songbirds, and we provide evidence that this is related to greenspace fragmentation.

Network integration modelling of feeder and BRT(bus rapid transit) to reduce the usage of private vehicles in Palembang’s suburban area

NASA Astrophysics Data System (ADS)

Nur'afalia, D.; Afifa, F.; Rubianto, L.; Handayeni, K. D. M. E.

2018-01-01

The aim of this research is to determine the optimal feeder network route that integrates with BRT (Bus Rapid Transit). Palembang, a high growing population city with unresolved transportation demand sector. BRT as main public transportation could not fulfill people’s demand in transportation, especially in Alang-Alang Lebar sub-district. As an impact, the usage of private vehicles increases along the movement toward the city center. The concept of Network Integration that integrates feeder network with BRT is expected to be a solution to suppress the rate of private vehicles’ usage and to improve public transportation service, so that the use of BRT will be increased in the suburban area of Palembang. The method used to identifying the optimal route using Route Analysis method is route analysis using Tranetsim 0.4. The best route is obtained based on 156 movement samples. The result is 58,7% from 199 mobility’s potency of private vehicle usage’s can be reduced if there is a feeder network’s route in Alang-Alang Lebar’s sub-district. From the result, the existance of integration between feeder network and BRT is potential enough to reduce the usage of private vehicles and supports the sustainability of transportation mobility in Palembang City.

Effect of stall design on dairy calf transition to voluntary feeding on an automatic milk feeder after introduction to group housing.

PubMed

Wilson, Tanya R; LeBlanc, Stephen J; DeVries, Trevor J; Haley, Derek B

2018-06-01

Automatic milk feeders (AMF) for young dairy calves are widely used in the dairy industry. These feeders are thought to have benefits for calf health and welfare and may reduce labor required for feeding; however, little is known about how calves adapt to feeding with AMF. The objective of this study was to observe the effects of feeding stall design on calves learning to use the AMF. The hypothesis was that solid side stalls, compared with steel bar stalls, would result in a longer latency to approach and feed from the AMF without assistance. A total of 147 Holstein calves (80 male and 67 female) were enrolled at 4 d of age, introduced to a group pen, and, at the same time, trained on an AMF. For training, calves were allowed to suck on the trainer's fingers and guided to the teat. Calves were allocated to 1 of 2 stall designs at the pen level, depending on which treatment cohort they were born into, either with steel bar stall walls (n = 46 male, 34 female calves) or with solid side stall walls (n = 34 male, 33 female calves). For 72 h after introductory training on the AMF, data from the feeders were collected and calf behavior was monitored by video. Outcomes measured included latency to first voluntary visit to the feeder and to first feeding, time spent in the feeder, amount of milk consumed over 72 h, number of retraining sessions required (retrained if <2 L was consumed every 12 h), and exploratory behavior, such as sniffing and licking of the feeder. Data were analyzed using mixed effects linear regression models or a Poisson model for the outcome of retraining. For certain outcomes the effects of stall design interacted with difficulty of training (willingness to enter feeder and drink); for the 38% of calves that were scored as moderately difficult to train on a scale of easy, moderate, or difficult, treatment (stall design) differences were detected. These calves took 2× longer to lick or bite toward the nipple, 2× longer to first voluntarily feeding, and consumed less milk over 72 h following training when trained on the steel bar stall design. These results suggest simple features of a stall may influence how quickly calves learn to use an AMF, but that the influence of stall wall design was affected by how easy calves were to train on the feeder upon initial introduction, which may depend in part on certain aspects of calf temperament. For many calves, solid side stalls at an AMF resulted faster in adaption than the steel bar stalls. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

View east, stone sluice, beginning of lower standing section, showing ...

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

View east, stone sluice, beginning of lower standing section, showing third drop, stone pier in center, cement piers to right - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

System Operations Studies : Feeder System Model. User's Manual.

DOT National Transportation Integrated Search

1982-11-01

The Feeder System Model (FSM) is one of the analytic models included in the System Operations Studies (SOS) software package developed for urban transit systems analysis. The objective of the model is to assign a proportion of the zone-to-zone travel...

Development of Experimental Setup of Metal Rapid Prototyping Machine using Selective Laser Sintering Technique

NASA Astrophysics Data System (ADS)

Patil, S. N.; Mulay, A. V.; Ahuja, B. B.

2018-04-01

Unlike in the traditional manufacturing processes, additive manufacturing as rapid prototyping, allows designers to produce parts that were previously considered too complex to make economically. The shift is taking place from plastic prototype to fully functional metallic parts by direct deposition of metallic powders as produced parts can be directly used for desired purpose. This work is directed towards the development of experimental setup of metal rapid prototyping machine using selective laser sintering and studies the various parameters, which plays important role in the metal rapid prototyping using SLS technique. The machine structure in mainly divided into three main categories namely, (1) Z-movement of bed and table, (2) X-Y movement arrangement for LASER movements and (3) feeder mechanism. Z-movement of bed is controlled by using lead screw, bevel gear pair and stepper motor, which will maintain the accuracy of layer thickness. X-Y movements are controlled using timing belt and stepper motors for precise movements of LASER source. Feeder mechanism is then developed to control uniformity of layer thickness metal powder. Simultaneously, the study is carried out for selection of material. Various types of metal powders can be used for metal RP as Single metal powder, mixture of two metals powder, and combination of metal and polymer powder. Conclusion leads to use of mixture of two metals powder to minimize the problems such as, balling effect and porosity. Developed System can be validated by conducting various experiments on manufactured part to check mechanical and metallurgical properties. After studying the results of these experiments, various process parameters as LASER properties (as power, speed etc.), and material properties (as grain size and structure etc.) will be optimized. This work is mainly focused on the design and development of cost effective experimental setup of metal rapid prototyping using SLS technique which will gives the feel of metal rapid prototyping process and its important parameters.

Cone-Beam Computed Tomography (CBCT) Hepatic Arteriography in Chemoembolization for Hepatocellular Carcinoma: Performance Depicting Tumors and Tumor Feeders

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

Lee, In Joon; Chung, Jin Wook, E-mail: chungjw@snu.ac.kr; Yin, Yong Hu

2015-10-15

PurposeThis study was designed to analyze retrospectively the performance of cone-beam computed tomography (CBCT) hepatic arteriography in depicting tumors and their feeders and to investigate the related determining factors in chemoembolization for hepatocellular carcinoma (HCC).MethodsEighty-six patients with 142 tumors satisfying the imaging diagnosis criteria of HCC were included in this study. The performance of CBCT hepatic arteriography for chemoembolization per tumor and per patient was evaluated using maximum intensity projection images alone (MIP analysis) or MIP combined with multiplanar reformation images (MIP + MPR analysis) regarding the following three aspects: tumor depiction, confidence of tumor feeder detection, and trackability of tumor feeders.more » Tumor size, tumor enhancement, tumor location, number of feeders, diaphragmatic motion, portal vein enhancement, and hepatic artery to parenchyma enhancement ratio were regarded as potential determining factors.ResultsTumors were depicted in 125 (88.0 %) and 142 tumors (100 %) on MIP and MIP + MPR analysis, respectively. Imaging performances on MIP and MIP + MPR analysis were good enough to perform subsegmental chemoembolization without additional angiographic investigation in 88 (62.0 %) and 128 tumors (90.1 %) on per-tumor basis and in 43 (50 %) and 73 (84.9 %) on per-patient basis, respectively. Significant determining factors for performance in MIP + MPR analysis on per tumor basis were tumor size (p = 0.030), tumor enhancement (0.005), tumor location (p = 0.001), and diaphragmatic motion (p < 0.001).ConclusionsCBCT hepatic arteriography provided sufficient information for subsegmental chemoembolization by depicting tumors and their feeders in the vast majority of patients. Combined analysis of MIP and MPR images was essential to enhance the performance of CBCT hepatic arteriography.« less

The Use of Filter-feeders to Manage Disease in a Changing World.

PubMed

Burge, Colleen A; Closek, Collin J; Friedman, Carolyn S; Groner, Maya L; Jenkins, Cody M; Shore-Maggio, Amanda; Welsh, Jennifer E

2016-10-01

Rapid environmental change is linked to increases in aquatic disease heightening the need to develop strategies to manage disease. Filter-feeding species are effective biofilters and can naturally mitigate disease risk to humans and wildlife. We review the role of filter-feeders, with an emphasis on bivalves, in altering disease outcomes via augmentation and reduction. Filtration can reduce transmission by removing pathogens from the water column via degradation and release of pathogens in pseudofeces. In other cases, filtration can increase pathogen transmission and disease risk. The effect of filtration on pathogen transmission depends on the selectivity of the filter-feeder, the degree of infectivity by the pathogen, the mechanism(s) of pathogen transmission and the ability of the pathogen to resist degradation. For example, some bacteria and viruses can resist degradation and accumulate within a filter-feeder leading to disease transmission to humans and other wildlife upon ingestion. Since bivalves can concentrate microorganisms, they are also useful as sentinels for the presence of pathogenic microorganisms. While somewhat less studied, other invertebrates, including ascidians and sponges may also provide ecosystem services by altering pathogen transmission. In all scenarios, climate change may affect the potential for filter-feeders to mitigate disease risk. We conclude that an assessment including empirical data and modeling of system-wide impacts should be conducted before selection of filter-feeders to mitigate disease. Such studies should consider physiology of the host and microbe and risk factors for negative impacts including augmentation of other pathogens. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Feeding group responses of a Neotropical termite assemblage to rain forest fragmentation.

PubMed

Davies, Richard G

2002-10-01

Biomass collapse and its associated microclimatic stresses within recently isolated rain forest fragments may negatively affect species diversity of most resident taxa. However, for some decomposer organisms, increased resource availability via accompanying tree die-off may effect positive responses, at least for a time, with implications for rates of nutrient cycling and greenhouse gas release. This study investigates the early effects of forest fragmentation on a Neotropical termite assemblage. Numbers of encounters (surrogate for relative abundance) and species richness of wood and leaf-litter feeders, soil feeders, and the whole assemblage, were studied across true forest islands and mainland sites at a hydroelectric reservoir in French Guiana. Results showed no overall effect of fragmentation on either total termite encounters or species richness. However, numbers of encounters and species richness of wood and leaf-litter feeders showed positive responses to forest fragmentation. By contrast, soil feeders showed a negative response for numbers of encounters and no significant effect for species richness. Environmental data suggest that increased tree die-off, and other edge effects associated with biomass collapse, were underway at the time of sampling. Resulting increase in resource availability may therefore explain the positive influence on wood and leaf-litter feeders. A possible decrease in predation pressure from ants with decrease in island size was not tested for, but was a likely effect of the flooded matrix habitat. Fragmentation effects on soil feeder encounters may be due to the energetic and microclimatic constraints of feeding lower down the humification gradient of termite food substrates, but were not sufficient to affect species richness. The patterns revealed suggest that rates of wood decomposition following tree die-off, and of soil nutrient cycling, under different rain forest fragmentation scenarios, merit further study.

A comparison of two systems for chlorinating water in rural Honduras.

PubMed

Henderson, Amy K; Sack, R Bradley; Toledo, Erick

2005-09-01

This study investigated a small subset of the two community water-disinfection systems--hypochlorinators and tablet feeders-in rural Honduras. Levels of residual chlorine were assessed at three locations within the distribution system: the tank, the proximal house, and the distal house. The levels of residual chlorine were compared with the standard guidelines set by the Pan American Health Organization and the International Rural Water Association for potable water that require a minimum of 1.0 (tank), 0.5 (proximal house), and 0.2 (distal house) ppm for each location. The levels of residual chlorine were also compared across systems, e.g. hypochlorinators to tablet feeders. At the tank and proximal house, tablet feeders had significantly higher mean values for levels of residual chlorine (measured in ppm) than hypochlorinators (tank: 1.20 vs 0.67; proximal house: 0.44 vs 0.32, p < 0.001 for both) with no significant difference at the distal house (0.16 vs 0.16). At the tank and proximal house, tablet feeders were more likely to meet recommended standards than hypochlorinators (90.3% vs 13.3%, p < 0.0001 and 41.3% vs 23.7%, p < 0.0001) with a smaller difference seen at the distal house (30.6% vs 27.1%, p = 0.24). The apparent dichotomy in chlorine levels of tablet feeders (e.g. between tank/proximal house and distal house) is discussed. The results suggest that tablet feeders may be more effective than hypochlorinators in supplying clean water in rural, resource-poor settings and possibly serve as an alternative technology for water disinfection. Further research on techniques for empowering and building capacity within community water boards will help organize and introduce sustainable water systems in developing countries.

GSM Web-Based Centralized Remote Wireless Automatic Controlling and Monitoring of Aquafeeder

NASA Astrophysics Data System (ADS)

Wong, C. L.; Idris, A.; Hasan, Z.

2016-03-01

This project is about producing a prototype to feed fishes at fish ponds of remote location with the use of GSM mobile phone. An automatic fish feeder is an electric device that has been designed to give out the right amount of pellets at the designed time. In this project, the automatic feeder designed consists of photovoltaic solar cells that are used to generate electricity and storing it into batteries. Solar charge controllers can be used to determine the rate of which current is drawn and added from the batteries. GSM cellular communication is used to allow user to control from a distance. Commands or instructions are sent to the operating system which in return runs the servomotor and blower by blowing certain amount of fish pallets into the pond to feed the fishes. The duration of the feeding processes is fixed by the user, hence the amount of fish food pallets released are precisely the same for each time. This technology is especially useful for fish farmers where they can remotely feed their fishes.

Container barge feeder service study : Bridgeport, New Haven, New London, Norwich

DOT National Transportation Integrated Search

2001-03-01

The Connecticut Department of Transportation (ConnDOT) has conducted this study to determine the need and opportunity for establishing a Container Barge Feeder Service along Long Island Sound between the Port of New York and New Jersey (NY&NJ) and th...

Evaluating the Magnitude and Duration of Cold Load Pick-up on Residential Distribution Feeders Using Multi-State Load Models

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

Schneider, Kevin P.; Sortomme, Eric; Venkata, S. S.

The increased level of demand that is associated with the restoration of service after an outage, Cold Load Pick-Up (CLPU), can be significantly higher than pre-outage levels, even exceeding the normal distribution feeder peak demand. These high levels of demand can delay restoration efforts and in extreme cases damage equipment. The negative impacts of CLPU can be mitigated with strategies that restore the feeder in sections, minimizing the load current. The challenge for utilities is to manage the current level on critical equipment while minimizing the time to restore service to all customers. Accurately modeling CLPU events is the firstmore » step in developing improved restoration strategies that minimize restoration times. This paper presents a new method for evaluating the magnitude of the CLPU peak, and its duration, using multi-state load models. The use of multi-state load models allows for a more accurate representation of the end-use loads that are present on residential distribution feeders.« less

Establishment of cell lines from adult T-cell leukemia cells dependent on negatively charged polymers.

PubMed

Kagami, Yoshitoyo; Uchiyama, Susumu; Kato, Harumi; Okada, Yasutaka; Seto, Masao; Kinoshita, Tomohiro

2017-07-05

Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L + HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L + HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L + HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts.

PubMed

Diecke, Sebastian; Lisowski, Leszek; Kooreman, Nigel G; Wu, Joseph C

2014-01-01

The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.

Analysis of the 60-Hz power system at KSC: The Orsino substation

NASA Technical Reports Server (NTRS)

Kalu, Alex O.

1989-01-01

An analysis of the Orsino Substation, a component (50 percent) of the 60-Hertz electric power system at the Kennedy Space Center, is presented. Presented here are separate single-line diagrams of the sixteen feeder circuits to permit easy access to information on the individual feeders for future planning. The load condition of each feeder and load break switch are presented and a heuristic reliability analysis of the system is performed. Information is given about the system fashion useful for decision making purposes. The beauty of it is in the simplified manner by which information about the system can be obtained.

Methods to Determine Recommended Feeder-Wide Advanced Inverter Settings for Improving Distribution System Performance

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

Rylander, Matthew; Reno, Matthew J.; Quiroz, Jimmy E.

This paper describes methods that a distribution engineer could use to determine advanced inverter settings to improve distribution system performance. These settings are for fixed power factor, volt-var, and volt-watt functionality. Depending on the level of detail that is desired, different methods are proposed to determine single settings applicable for all advanced inverters on a feeder or unique settings for each individual inverter. Seven distinctly different utility distribution feeders are analyzed to simulate the potential benefit in terms of hosting capacity, system losses, and reactive power attained with each method to determine the advanced inverter settings.

An automatic 14-day paste diet feeder for animals

NASA Technical Reports Server (NTRS)

Vasques, Marilyn; Mulenburg, Jerry; Gundo, Dan; Griffith, Jon

1994-01-01

During a centrifuge experiment, any interruption that requires stopping the centrifuge may influence the results. Centrifuges often must be stopped for animal maintenance (food, water and waste removal), especially in cases of timed feedings. To eliminate the need for stopping the centrifuge while still providing timed feeding, an automatic paste diet feeder was developed. The feeder is based on a constant volume concept and can deliver a predetermined amount of paste diet at specified time intervals. This unit was supported by water delivery and waste collection systems. The entire system performed reliably and maintained the animals well for a continuous centrifugation experiment of 14 days.

Systems Operation Studies for Automated Guideway Transit Systems: Feeder Systems Model Functional Specification

DOT National Transportation Integrated Search

1981-01-01

This document specifies the functional requirements for the AGT-SOS Feeder Systems Model (FSM), the type of hardware required, and the modeling techniques employed by the FSM. The objective of the FSM is to map the zone-to-zone transit patronage dema...

View southeast, stone sluice, top of lower standing section, showing ...

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

View southeast, stone sluice, top of lower standing section, showing row of cement piers in center, retaining wall to left, barge canal sluice to right - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

47. FEEDER CANAL AT WILL'S BASIN. BOATS IN THE FOREGROUND ...

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

47. FEEDER CANAL AT WILL'S BASIN. BOATS IN THE FOREGROUND SHOW THE CONTRAST BETWEEN A FULLY LOADED CANAL BOAT (LEFT) AND AN EMPTY ONE (RIGHT). D, L & W RAILROAD'S DOUBLE INTERSECTION PRATT TRUSS BRIDGE IS VISIBLE IN BACKGROUND. - Morris Canal, Phillipsburg, Warren County, NJ

34. INTERIOR VIEW, SAME AS ABOVE WITH THE FEEDER ROD ...

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

34. INTERIOR VIEW, SAME AS ABOVE WITH THE FEEDER ROD BEING OFFERED INTO THE MACHINE; UNLIKE THE OTHER NAIL CUTTING MACHINES, THE NAIL PLATE MUST BE HAND FLIPPED AND HAND FED WITH THIS MACHINE - LaBelle Iron Works, Thirtieth & Wood Streets, Wheeling, Ohio County, WV

Termites as bioindicators of habitat quality in the caatinga, Brazil: is there agreement between structural habitat variables and the sampled assemblages?

PubMed

Alves, W de F; Mota, A S; Lima, R A A de; Bellezoni, R; Vasconcellos, A

2011-01-01

The composition of termite assemblages was analyzed in three caatinga sites of the Estação Ecológica do Seridó, located in the municipality of Serra Negra do Norte, in the state of Rio Grande do Norte, Brazil. These sites have been subjected to selective logging, and cleared for pasture and farming. A standardized sampling protocol for termite assemblages (30h/person/site) was conducted between September 2007 and February 2009. At each site we measured environmental variables, such as soil pH and organic matter, necromass stock, vegetation height, stem diameter at ankle height (DAH) and the largest and the smallest crown width. Ten species of termites, belonging to eight genera and three families, were found at the three experimental sites. Four feeding groups were sampled: wood-feeders, soil-feeders, wood-soil interface feeders and leaf-feeders. The wood-feeders were dominant in number of species and number of encounters at all sites. In general, the sites were not significantly different in relation to the environmental variables measured. The same pattern was observed for termite assemblages, where no significant differences in species richness, relative abundance and taxonomic and functional composition were observed between the three sites. The agreement between composition of assemblages and environmental variables reinforces the potential of termites as biological indicators of habitat quality.

Four eyes match better than two: Sharing of precise patch-use time among socially foraging domestic chicks.

PubMed

Xin, Qiuhong; Ogura, Yukiko; Matsushima, Toshiya

2017-07-01

To examine how resource competition contributes to patch-use behaviour, we examined domestic chicks foraging in an I-shaped maze equipped with two terminal feeders. In a variable interval schedule, one feeder supplied grains three times more frequently than the other, and the sides were reversed midway through the experiment. The maze was partitioned into two lanes by a transparent wall, so that chicks fictitiously competed without actual interference. Stay time at feeders was compared among three groups. The "single" group contained control chicks; the "pair" group comprised the pairs of chicks tested in the fictitious competition; "mirror" included single chicks accompanied by their respective mirror images. Both "pair" and "mirror" chicks showed facilitated running. In terms of the patch-use ratio, "pair" chicks showed precise matching at approximately 3:1 with significant mutual dependence, whereas "single" and "mirror" chicks showed a comparable under-matching. The facilitated running increased visits to feeders, but failed to predict the patch-use ratio of the subject. At the reversal, quick switching occurred similarly in all groups, but the "pair" chicks revealed a stronger memory-based matching. Perceived competition therefore contributes to precise matching and lasting memory of the better feeder, in a manner dissociated from socially facilitated food search. Copyright © 2017 Elsevier B.V. All rights reserved.

Response of a benthic suspension feeder ( Crassostrea virginica Gmelin) to three centuries of anthropogenic eutrophication in Chesapeake Bay

NASA Astrophysics Data System (ADS)

Kirby, Michael X.; Miller, Henry M.

2005-03-01

Biogenic reefs built by oysters and other suspension feeders are vital components of estuarine ecosystems. By consuming phytoplankton, suspension feeders act to suppress accumulation of organic matter in the water column. Nutrient loading increases the rate of primary production, thereby causing eutrophication. As suspension feeders consume more organic matter from increasing abundance of phytoplankton, their rate of growth should also increase if they are food limited. We show here that the eastern oyster, Crassostrea virginica (Gmelin), from St. Mary's and Patuxent rivers, Chesapeake Bay, grew faster during anthropogenic eutrophication relative to C. virginica before eutrophication. Growth of shell height, shell thickness and adductor muscle increased after eutrophication began in the late 18th century. After 1860, growth decreased, perhaps reflecting the negative effects of hypoxia, harmful algal blooms, disease and fishing on oyster growth. These results are consistent with the view that an increasing supply of phytoplankton resulting from eutrophication enhanced growth of C. virginica between 1760 and 1860, before oyster reefs were degraded by destructive fishing practices between 1870 and 1930. Alternative factors, such as changes in water temperature, salinity, and fishing are less likely to be responsible for this pattern. These results have implications for restoration of oyster reefs in order to mitigate the effects of eutrophication in estuaries, as well as the paleoecological relationship between suspension feeders and paleoproductivity.

View north, stone sluice, head of 40foot break, showing failed ...

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

View north, stone sluice, head of 40-foot break, showing failed arch in center, stone pier in center right, cement piers to upper left, retaining wall in background - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

Dry piston coal feeder

DOEpatents

Hathaway, Thomas J.; Bell, Jr., Harold S.

1979-01-01

This invention provides a solids feeder for feeding dry coal to a pressurized gasifier at elevated temperatures substantially without losing gas from the gasifier by providing a lock having a double-acting piston that feeds the coals into the gasifier, traps the gas from escaping, and expels the trapped gas back into the gasifier.

76 FR 3655 - Bunker Hill Groundwater Basin, Riverside-Corona Feeder Project, San Bernardino and Riverside...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-20

... DEPARTMENT OF THE INTERIOR Bureau of Reclamation Bunker Hill Groundwater Basin, Riverside-Corona... Draft Environmental Impact Statement (SDEIR/DEIS) for the proposed Riverside-Corona Feeder (RCF) Project... Bernardino, California 92410 Corona Public Library, 650 South Main Street, Corona, California 92882 Riverside...

30 CFR 57.12086 - Location of trolley wire.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Location of trolley wire. 57.12086 Section 57... Underground Only § 57.12086 Location of trolley wire. Trolley and trolley feeder wire shall be installed... limitations would prevent the safe installation or use of such trolley and trolley feeder wire. ...

30 CFR 57.12086 - Location of trolley wire.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Location of trolley wire. 57.12086 Section 57... Underground Only § 57.12086 Location of trolley wire. Trolley and trolley feeder wire shall be installed... limitations would prevent the safe installation or use of such trolley and trolley feeder wire. ...

Optimal foraging by birds: feeder-based experiments for secondary and post-secondary students

USDA-ARS?s Scientific Manuscript database

Optimal foraging theory attempts to explain the foraging patterns observed in animals, including their choice of particular food items and foraging locations. Here, we describe three exercises designed to test hypotheses about food choice and foraging habitat preference using bird feeders. These e...

Theoretical Comparison of Fixed Route Bus and Flexible Route Subscription Bus Feeder Service in Low Density Areas

DOT National Transportation Integrated Search

1975-03-01

parametric variation of demand density was used to compare service level and cost of two alternative systems for providing low density feeder service. Supply models for fixed route and flexible route service were developed and applied to determine ra...

77 FR 13191 - Airworthiness Directives; Bombardier, Inc. Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-06

... corrosion and inadequate silver-plating. This AD requires replacing ADG power feeder cables. We are issuing this AD to prevent galvanic corrosion on ADG power feeder cables, which could result in damage to the... damaged due to galvanic corrosion. It was subsequently determined that the silver- plating is inadequate...

Novel duplex vapor: Electrochemical method for silicon solar cells. [chemical reactor for a silicon sodium reaction system

NASA Technical Reports Server (NTRS)

Nanis, L.; Sanjurjo, A.; Sancier, K.

1979-01-01

The scaled up chemical reactor for a SiF4-Na reaction system is examined for increased reaction rate and production rate. The reaction system which now produces 5 kg batches of mixed Si and NaF is evaluated. The reactor design is described along with an analysis of the increased capacity of the Na chip feeder. The reactor procedure is discussed and Si coalescence in the reaction products is diagnosed.

Cancer Immunology in an Inducible Model of Breast Cancer

DTIC Science & Technology

2005-04-01

mutations or deletions that allow the binding Feeder cells were prepared from 12-14-day-old C57B16 of synthetic hormone analogues to the HBDs, but not of...transduction efficiency seen in the presence (37%, v/v) were added, and samples were incubated for of the hormone analogue , but rather the fraction of 10...cm 2 by the and electroporated (BioRad gene pulser; 960 jF, 300 V) chloroquine /calcium phosphate method, as described with 4 jig of the NotI

Preoperative catheter spinal angiography and embolization of cervical spinal tumors: Outcomes from a single center

PubMed Central

Leng, Lewis Z; Kimball, David; Marcus, Joshua; Knopman, Jared; Laufer, Ilya; Bilsky, Mark; Gobin, Y Pierre

2016-01-01

Objective The existing literature regarding preoperative cervical spinal tumor embolization is sparse, with few discussions on the indications, risks, and best techniques. We present our experience with the preoperative endovascular management of hypervascular cervical spinal tumors. Methods We performed a retrospective review of all patients who underwent preoperative spinal angiography (regardless of whether tumor embolization was performed) at our institution (from 2002 to 2012) for primary and metastatic cervical spinal tumors. Tumor vascularity was graded from 0 (tumor blush equal to the normal adjacent vertebral body) to 3 (intense tumor blush with arteriovenous shunting). Tumors were considered “hypervascular” if they had a tumor vascular grade from 1 to 3. Embolic materials included particles, liquid embolics, and detachable coils. The main embolization technique was superselective catheterization of an arterial tumor feeder followed by injection of embolic material. This technique could be used alone or supplemented with occlusion of dangerous anastomoses of the vertebral artery as needed to prevent inadvertent embolization of the vertebrobasilar system. In cases when superselective catheterization of the tumoral feeder was not feasible, embolization was performed from a proximal catheter position after occlusion of branches supplying areas other than the tumor (“flow diversion”). Results A total of 47 patients with 49 cervical spinal tumors were included in this study. Of the 49 total tumors, 41 demonstrated increased vascularity (vascularity score > 0). The most common tumor pathology in our series was renal cell carcinoma (RCC) (N = 16; 32.7% of all tumors) followed by thyroid carcinoma (N = 7; 14.3% of all tumors). Tumor embolization was undertaken in 25 hypervascular tumors resulting in complete, near-complete, and partial embolization in 36.0% (N = 9), 44.0% (N = 11), and 20.0% (N = 5) of embolized tumors, respectively. We embolized 42 tumor feeders in 25 tumors. The most commonly embolized tumor feeders were branches of the vertebral artery (19.0%; N = 8), the deep cervical artery (19.0%; N = 8), and the ascending cervical artery (19.0%; N = 8). Sixteen hypervascular tumors were not embolized because of minimal hypervascularity (8/16), unacceptably high risk of spinal cord or vertebrobasilar ischemia (4/16), failed superselective catheterization of tumor feeder (3/16), and cancellation of surgery (1/16). Vertebral artery occlusion was performed in 20% of embolizations. There were no new post-procedure neurological deficits or any serious adverse events. Estimated blood loss data from this cohort show a significant decrease in operative blood loss for embolized tumors of moderate and significant hypervascularity. Conclusions Preoperative embolization of cervical spinal tumors can be performed safely and effectively in centers with significant experience and a standardized approach. PMID:27020696

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

PubMed

Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

2017-10-01

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56 + CD3 - ) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO ™ 10, CellGro ® , TexMACS ™ , and NK MACS ® ). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56 + CD3 - target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56 dim CD16 pos and CD56 bright CD16 dim&neg NK subsets on day 14 with cells cultivated in NK MACS ® media. Moreover, effector cell expansion in manually performed experiments with NK MACS ® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123 pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara).

PubMed

Guiderdoni, E; Chaïr, H

1992-11-01

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.

78 FR 79338 - Airworthiness Directives; Bombardier, Inc. Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-30

...We propose to adopt a new airworthiness directive (AD) for certain Bombardier, Inc. Model DHC-8-400 series airplanes. This proposed AD was prompted by reports of missing clamps that are required to provide positive separation between the alternating current (AC) feeder cables and the hydraulic line of the landing gear alternate extension. This proposed AD would require inspecting for missing clamps, and related investigative and corrective actions if necessary. We are proposing this AD to detect and correct chafing of the AC feeder cable. A chafed and arcing AC feeder cable could puncture the adjacent hydraulic line, which, in combination with the use of the alternate extension system, could result in an in-flight fire.

Technologies to Increase PV Hosting Capacity in Distribution Feeders: Preprint

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

Ding, Fei; Mather, Barry; Gotseff, Peter

This paper studies the distributed photovoltaic (PV) hosting capacity in distribution feeders by using the stochastic analysis approach. Multiple scenario simulations are conducted to analyze several factors that affect PV hosting capacity, including the existence of voltage regulator, PV location, the power factor of PV inverter and Volt/VAR control. Based on the conclusions obtained from simulation results, three approaches are then proposed to increase distributed PV hosting capacity, which can be formulated as the optimization problem to obtain the optimal solution. All technologies investigated in this paper utilize only existing assets in the feeder and therefore are implementable for amore » low cost. Additionally, the tool developed for these studies is described.« less

Technologies to Increase PV Hosting Capacity in Distribution Feeders

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

Ding, Fei; Mather, Barry; Gotseff, Peter

This paper studies the distributed photovoltaic (PV) hosting capacity in distribution feeders by using the stochastic analysis approach. Multiple scenario simulations are conducted to analyze several factors that affect PV hosting capacity, including the existence of voltage regulator, PV location, the power factor of PV inverter and Volt/VAR control. Based on the conclusions obtained from simulation results, three approaches are then proposed to increase distributed PV hosting capacity, which can be formulated as the optimization problem to obtain the optimal solution. All technologies investigated in this paper utilize only existing assets in the feeder and therefore are implementable for amore » low cost. Additionally, the tool developed for these studies is described.« less

Petrological constraints on the recycling of mafic crystal mushes, magma ascent and intrusion of braided sills in the Torres del Paine mafic complex (Patagonia)

NASA Astrophysics Data System (ADS)

Leuthold, Julien; Müntener, Othmar; Baumgartner, Lukas; Putlitz, Benita

2014-05-01

Cumulate and crystal mush disruption and reactivation are difficult to recognise in coarse grained shallow plutonic rocks. Mafic minerals included in hornblende and zoned plagioclase provide snapshots of early crystallization and cumulate formation, but are difficult to interpret in terms of the dynamics of magma ascent and possible links between silicic and mafic rock emplacement. We will present the field relations, the microtextures and the mineral chemistry of the Miocene mafic sill complex of the Torres del Paine intrusive complex (Patagonia, Chile) and its sub-vertical feeder-zone. The mafic sill complex was built up by a succession of braided sills of shoshonitic and high-K calc-alkaline porphyritic hornblende-gabbro and fine grained monzodioritic sills. The mafic units were over-accreted over 41±11 ka, underplating the overlying granite. Local diapiric structures and felsic magma accumulation between sills indicate limited separation of intercumulus liquid from the mafic sills. Anhedral hornblende cores, with olivine + clinopyroxene ± plagioclase ± apatite inclusions, crystallized at temperatures >900°C and pressures of ~300 to ~500 MPa. The corresponding rims and monzodiorite matrix crystallized at <830°C, ~70 MPa. This abrupt compositional variation suggests stability and instability of hornblende during mafic roots recycling and subsequent decompression. The near lack of intercumulus crystals in the sub-vertical feeder zone layered gabbronorite and pyroxene-hornblende gabbronorite stocks testifies that melt is more efficiently extracted than in sills, resulting in a cumulate signature in the feeding system. The emplacement age of the sill complex topmost granitic unit is identical, within uncertainties, to the feeder zone mafic cumulates. Granitic liquids formed by AFC processes and were extracted at high temperature (T>950°C) from the middle crust reservoir to the emplacement level. We show that hornblende-plagioclase thermobarometry is a useful monitor for the determination of segregation conditions of granitic magmas from gabbroic crystal mushes, and for monitoring the evolution of shallow crustal magmatic crystallization, decompression and cooling.

Distribution of free gas and 3D mirror image structures beneath Sevastopol mud volcano, Black sea, from 3D high resolution wide-angle seismic data

NASA Astrophysics Data System (ADS)

Krabbenhoeft, A.; Papenberg, C. A.; Klaeschen, D.; Bialas, J.

2016-12-01

The goal of this study is to image the sub-seafloor structure beneath the Sevastopol mud volcano (SMV), Sorokin Trough, SE of the Crimean peninsula, Black Sea. The focus lies on structures of/within the feeder channel, the distribution of gas and gas hydrates, and their relation to fluid migration zones in sediments. This study concentrates on a 3D high resolution seismic grid (7 km x 2.5 km) recorded with 13 ocean bottom stations (OBS). The 3D nature of the experiment results from the geometry of 68 densely spaced (25/50 m) profiles, as well as the cubical configuration of the densely spaced receivers on the seafloor ( 300 m station spacing). The seismic profiles are typically longer than 6 km which results in large offsets for the reflections of the OBS. This enables the study of the seismic velocities of the sub-seafloor sediments and additionally large offset incident analysis.The 3D Kirchhoff mirror image time migration, applied to all OBS sections including all shots from all profiles, leads to a spatial image of the sub-seafloor. Here, the migration was applied with the velocity distribution of 1.49 km/s in the water column, 1.5 km/s below the seafloor (bsf) increasing to 2 km/s for the deeper sediments at 2 s bsf. Acoustic blanking occurs beneath the south-easterly located OBS and is associated with the feeder channel of the mud volcano. There, gas from depth can vertically migrate to the seafloor and on its way to the surface horizontally distribute patchily within sediment layers. High amplitude reflections are not observed as continuous reflections, but in a patchy distribution. They are associated with accumulations of gas. Also structures exist within the feeder channel of the SMV.3D mirror imaging proves to be a good tool to seismically image structures compared with 2D streamer seismics, especially steep dipping reflectors and structures which are otherwise obscured by signal scattering, i.e structures associated with fluid migration paths.

Splashing, feeding, contracting: Drop impact and fluid dynamics of Vorticella

NASA Astrophysics Data System (ADS)

Pepper, Rachel E.

This thesis comprises two main topics: understanding drop impact and splashing, and studying the feeding and contracting of the microorganism Vorticella. In Chapter 1, we study the effect of substrate compliance on the splash threshold of a liquid drop using an elastic membrane under variable tension. We find that splashing can be suppressed by reducing this tension. Measurements of the velocity and acceleration of the spreading drop after impact indicate that the splashing behavior is set at very early times after, or possibly just before, impact, far before the actual splash occurs. We also provide a model for the tension dependence of the splashing threshold. In Chapter 2, we study the evolution of the ejected liquid sheet, or lamella, created after impact of a liquid drop onto a solid surface using high-speed video. We find that the lamella rim thickness is always much larger than the boundary layer thickness, and that this thickness decreases with increasing impact speed. We also observe an unusual plateau behavior in thickness versus time at higher impact speeds as we approach the splash threshold. In Chapter 3, we show through calculations, simulations, and experiments that the eddies often observed near sessile filter feeders are due to the presence of nearby boundaries. We model the common filter feeder Vorticella, and also track particles around live feeding Vorticella to determine the experimental flow field. Our models are in good agreement both with each other and with the experiments. We also provide simple approximate equations to predict experimental eddy sizes due to boundaries. In Chapter 4, we show through calculations that filter feeders such as Vorticella can greatly enhance their nutrient uptake by feeding at an angle rather than perpendicular to a substrate. We also show experimental evidence that living Vorticella use this strategy. Finally, in Chapter 5, we discuss possible future directions for these projects, including potential insights from a close examination of lamella behavior at the splash threshold, and calculations to determine if Vorticella contract rapidly towards the substrate to which they are attached in order to mix the surrounding fluid.

46 CFR 58.25-55 - Overcurrent protection for steering-gear systems.

Code of Federal Regulations, 2010 CFR

2010-10-01

... overcurrent protection except short-circuit protection that is instantaneous and rated at 400% to 500% of— (1... for steering-gear systems. (a) Each feeder circuit for steering must be protected by a circuit breaker... motor for an alternating-current motor. (b) No feeder circuit for steering may have any overcurrent...

26 CFR 1.1402(a)-14 - Options available to farmers in computing net earnings from self-employment for taxable years...

Code of Federal Regulations, 2013 CFR

2013-04-01

... held for breeding or dairy purposes, and $600 from the sale of a tractor. The income from the sale of... the sale of feeder cattle, which C bought for $500. The income from the sale of the feeder cattle is...

26 CFR 1.1402(a)-14 - Options available to farmers in computing net earnings from self-employment for taxable years...

Code of Federal Regulations, 2011 CFR

2011-04-01

... held for breeding or dairy purposes, and $600 from the sale of a tractor. The income from the sale of... the sale of feeder cattle, which C bought for $500. The income from the sale of the feeder cattle is...

26 CFR 1.1402(a)-14 - Options available to farmers in computing net earnings from self-employment for taxable years...

Code of Federal Regulations, 2014 CFR

2014-04-01

... held for breeding or dairy purposes, and $600 from the sale of a tractor. The income from the sale of... the sale of feeder cattle, which C bought for $500. The income from the sale of the feeder cattle is...

26 CFR 1.1402(a)-14 - Options available to farmers in computing net earnings from self-employment for taxable years...

Code of Federal Regulations, 2012 CFR

2012-04-01

... held for breeding or dairy purposes, and $600 from the sale of a tractor. The income from the sale of... the sale of feeder cattle, which C bought for $500. The income from the sale of the feeder cattle is...

View southeast, stone sluice, top of upper standing section, showing ...

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

View southeast, stone sluice, top of upper standing section, showing cement piers over arch in foreground, foot of break at third drop in center, retaining wall to left, barge canal sluice to right - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

77 FR 70355 - Airworthiness Directives; The Boeing Company Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-26

... leakage zone) or heat damage to the APU power feeder cable, insulation blankets, or pressure bulkhead...) of the NPRM requires repair of the APU power feeder, insulation blankets, and clamps, if no primer... bulletin, which states, ``If visual indications of heat damage are found, do steps 6.c through 6.f...

30 CFR 77.1800 - Cutout switches.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Cutout switches. 77.1800 Section 77.1800... Wires and Trolley Feeder Wires § 77.1800 Cutout switches. Trolley wires and trolley feeder wires shall be provided with cutout switches at intervals of not more than 2,000 feet and near the beginning of...

30 CFR 77.1800 - Cutout switches.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Cutout switches. 77.1800 Section 77.1800... Wires and Trolley Feeder Wires § 77.1800 Cutout switches. Trolley wires and trolley feeder wires shall be provided with cutout switches at intervals of not more than 2,000 feet and near the beginning of...

30 CFR 77.1800 - Cutout switches.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Cutout switches. 77.1800 Section 77.1800... Wires and Trolley Feeder Wires § 77.1800 Cutout switches. Trolley wires and trolley feeder wires shall be provided with cutout switches at intervals of not more than 2,000 feet and near the beginning of...

30 CFR 77.1800 - Cutout switches.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Cutout switches. 77.1800 Section 77.1800... Wires and Trolley Feeder Wires § 77.1800 Cutout switches. Trolley wires and trolley feeder wires shall be provided with cutout switches at intervals of not more than 2,000 feet and near the beginning of...

46 CFR 58.25-65 - Feeder circuits.

Code of Federal Regulations, 2013 CFR

2013-10-01

... seconds of loss of power from the vessel's service switchboard; (2) Comes from an independent source of power in the steering-gear compartment; (3) Is used for no other purpose; and (4) Has a capacity for one... feeder circuit must have a current-carrying capacity of— (1) 125% of the rated full-load current rating...

46 CFR 58.25-65 - Feeder circuits.

Code of Federal Regulations, 2011 CFR

2011-10-01

... seconds of loss of power from the vessel's service switchboard; (2) Comes from an independent source of power in the steering-gear compartment; (3) Is used for no other purpose; and (4) Has a capacity for one... feeder circuit must have a current-carrying capacity of— (1) 125% of the rated full-load current rating...

46 CFR 58.25-65 - Feeder circuits.

Code of Federal Regulations, 2014 CFR

2014-10-01

... seconds of loss of power from the vessel's service switchboard; (2) Comes from an independent source of power in the steering-gear compartment; (3) Is used for no other purpose; and (4) Has a capacity for one... feeder circuit must have a current-carrying capacity of— (1) 125% of the rated full-load current rating...

46 CFR 58.25-65 - Feeder circuits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... seconds of loss of power from the vessel's service switchboard; (2) Comes from an independent source of power in the steering-gear compartment; (3) Is used for no other purpose; and (4) Has a capacity for one... feeder circuit must have a current-carrying capacity of— (1) 125% of the rated full-load current rating...

46 CFR 58.25-65 - Feeder circuits.

Code of Federal Regulations, 2010 CFR

2010-10-01

... seconds of loss of power from the vessel's service switchboard; (2) Comes from an independent source of power in the steering-gear compartment; (3) Is used for no other purpose; and (4) Has a capacity for one... feeder circuit must have a current-carrying capacity of— (1) 125% of the rated full-load current rating...

78 FR 35314 - Availability of Final Environmental Impact Statement; Bunker Hill Groundwater Basin, Riverside...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-12

... DEPARTMENT OF THE INTERIOR Bureau of Reclamation [A10-1999-6000-100-00-0-0-3, 3501000] Availability of Final Environmental Impact Statement; Bunker Hill Groundwater Basin, Riverside-Corona Feeder... proposed Riverside-Corona Feeder Project. DATES: The Bureau of Reclamation will not make a decision on the...

Counseling in the Elementary Feeder Schools.

ERIC Educational Resources Information Center

Dunham, Virginia

This brief paper presents the concept of transition counseling between a junior high school and its feeder school(s), designed to make the change from elementary into junior high less traumatic. Aside from routine sixth grade counseling, the counselors expanded their base of counseling to include all types of problems as well as all grade levels.…

40 CFR Table 4 to Subpart Kkkkk of... - Requirements for Performance Tests

Code of Federal Regulations, 2010 CFR

2010-07-01

... block average pressure drop values for the three test runs, and determine and record the 3-hour block... limit for the limestone feeder setting Data from the limestone feeder during the performance test You must ensure that you maintain an adequate amount of limestone in the limestone hopper, storage bin...

Energy Systems Integration News | Energy Systems Integration Facility |

Science.gov Websites

distribution feeder models for use in hardware-in-the-loop (HIL) experiments. Using this method, a full feeder ; proposes an additional control loop to improve frequency support while ensuring stable operation. The and Frequency Deviation," also proposes an additional control loop, this time to smooth the wind

Exploring associations between international trade and environmental factors with establishment patterns of exotic Scolytinae

Treesearch

Lorenzo Marini; Robert A. Haack; Robert J. Rabaglia; Edoardo Petrucco Toffolo; Andrea Battisti; Massimo Faccoli

2011-01-01

Although invasion of exotic ambrosia beetles (fungus feeders) and bark beetles (phloem feeders) (Coleoptera: Curculionidae: Scolytinae) is considered a major threat to forest health worldwide, no studies have quantitatively investigated the anthropogenic and environmental factors shaping the biogeographical patterns of invasion by these insects across large spatial...

Blood feeding behavior of the stable fly

USDA-ARS?s Scientific Manuscript database

Stable fly is a fly that looks similar to a house fly but both sexes are blood feeders. Blood is required for successful fertilization and development of eggs. Bites are painful but there is usually no pain after the fly stops feeding. The stable fly is a persistent feeder and will continue trying t...

75 FR 78758 - Prohibited Transaction Exemptions From Certain Prohibited Transaction Restrictions

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-16

..., between the DB Torus Japan Master Portfolio (the Master Fund), in which the assets of a client employee...) Prior to investing in DB Torus Japan Fund Ltd. (hereinafter the Feeder Fund, the vehicle through which... DB Torus Japan Fund Ltd. (hereinafter the Feeder Fund, the vehicle through which investments in the...

Coal gasification system with a modulated on/off control system

DOEpatents

Fasching, George E.

1984-01-01

A modulated control system is provided for improving regulation of the bed level in a fixed-bed coal gasifier into which coal is fed from a rotary coal feeder. A nuclear bed level gauge using a cobalt source and an ion chamber detector is used to detect the coal bed level in the gasifier. The detector signal is compared to a bed level set point signal in a primary controller which operates in proportional/integral modes to produce an error signal. The error signal is modulated by the injection of a triangular wave signal of a frequency of about 0.0004 Hz and an amplitude of about 80% of the primary deadband. The modulated error signal is fed to a triple-deadband secondary controller which jogs the coal feeder speed up or down by on/off control of a feeder speed change driver such that the gasifier bed level is driven toward the set point while preventing excessive cycling (oscillation) common in on/off mode automatic controllers of this type. Regulation of the bed level is achieved without excessive feeder speed control jogging.

Improvement of automatic fish feeder machine design

NASA Astrophysics Data System (ADS)

Chui Wei, How; Salleh, S. M.; Ezree, Abdullah Mohd; Zaman, I.; Hatta, M. H.; Zain, B. A. Md; Mahzan, S.; Rahman, M. N. A.; Mahmud, W. A. W.

2017-10-01

Nation Plan of action for management of fishing is target to achieve an efficient, equitable and transparent management of fishing capacity in marine capture fisheries by 2018. However, several factors influence the fishery production and efficiency of marine system such as automatic fish feeder machine could be taken in consideration. Two latest fish feeder machines have been chosen as the reference for this study. Based on the observation, it has found that the both machine was made with heavy structure, low water and temperature resistance materials. This research’s objective is to develop the automatic feeder machine to increase the efficiency of fish feeding. The experiment has conducted to testing the new design of machine. The new machine with maximum storage of 5 kg and functioning with two DC motors. This machine able to distribute 500 grams of pellets within 90 seconds and longest distance of 4.7 meter. The higher speed could reduce time needed and increase the distance as well. The minimum speed range for both motor is 110 and 120 with same full speed range of 255.

Mathematical analysis of the honeybee waggle dance.

PubMed

Okada, R; Ikeno, H; Kimura, T; Ohashi, Mizue; Aonuma, H; Ito, E

2012-01-01

A honeybee informs her nestmates of the location of a flower by doing a waggle dance. The waggle dance encodes both the direction of and distance to the flower from the hive. To reveal how the waggle dance benefits the colony, we created a Markov model of bee foraging behavior and performed simulation experiments by incorporating the biological parameters that we obtained from our own observations of real bees as well as from the literature. When two feeders were each placed 400 m away from the hive in different directions, a virtual colony in which honeybees danced and correctly transferred information (a normal, real bee colony) made significantly greater numbers of successful visits to the feeders compared to a colony with inaccurate information transfer. Howerer, when five feeders were each located 400 m from the hive, the inaccurate information transfer colony performed better than the normal colony. These results suggest that dancing's ability to communicate accurate information depends on the number of feeders. Furthermore, because non-dancing colonies always made significantly fewer visits than those two colonies, we concluded that dancing behavior is beneficial for hives' ability to visit food sources.

Preliminary analysis of hub and spoke air freight distribution system

NASA Technical Reports Server (NTRS)

Whitehead, A. H., Jr.

1978-01-01

A brief analysis is made of the hub and spoke air freight distribution system which would employ less than 15 hub centers world wide with very large advanced distributed-load freighters providing the line-haul delivery between hubs. This system is compared to a more conventional network using conventionally-designed long-haul freighters which travel between numerous major airports. The analysis calculates all of the transportation costs, including handling charges and pickup and delivery costs. The results show that the economics of the hub/spoke system are severely compromised by the extensive use of feeder aircraft to deliver cargo into and from the large freighter terminals. Not only are the higher costs for the smaller feeder airplanes disadvantageous, but their use implies an additional exchange of cargo between modes compared to truck delivery. The conventional system uses far fewer feeder airplanes, and in many cases, none at all. When feeder aircraft are eliminated from the hub/spoke system, however, that system is universally more economical than any conventional system employing smaller line-haul aircraft.

Alternatives to the 15% Rule

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

Broderick, Robert Joseph; Quiroz, Jimmy Edward; Reno, Matthew J.

2015-11-01

The third solicitation of the California Solar Initiative (CSI) Research, Development, Demonstration and Deployment (RD&D) Program established by the California Public Utility Commission (CPUC) is supporting the Electric Power Research Institute (EPRI), National Renewable Energy Laboratory (NREL), and Sandia National Laboratories (SNL) with collaboration from Pacific Gas and Electric (PG&E), Southern California Edison (SCE), and San Diego Gas and Electric (SDG&E), in research to improve the Utility Application Review and Approval process for interconnecting distributed energy resources to the distribution system. Currently this process is the most time - consuming of any step on the path to generating power onmore » the distribution system. This CSI RD&D solicitation three project has completed the tasks of collecting data from the three utilities, clustering feeder characteristic data to attain representative feeders, detailed modeling of 16 representative feeders, analysis of PV impacts to those feeders, refinement of current screening processes, and validation of those suggested refinements. In this report each task is summarized to produce a final summary of all components of the overall project.« less

Likeability of Garden Birds: Importance of Species Knowledge & Richness in Connecting People to Nature.

PubMed

Cox, Daniel T C; Gaston, Kevin J

2015-01-01

Interacting with nature is widely recognised as providing many health and well-being benefits. As people live increasingly urbanised lifestyles, the provision of food for garden birds may create a vital link for connecting people to nature and enabling them to access these benefits. However, it is not clear which factors determine the pleasure that people receive from watching birds at their feeders. These may be dependent on the species that are present, the abundance of individuals and the species richness of birds around the feeders. We quantitatively surveyed urban households from towns in southern England to determine the factors that influence the likeability of 14 common garden bird species, and to assess whether people prefer to see a greater abundance of individuals or increased species richness at their feeders. There was substantial variation in likeability across species, with songbirds being preferred over non-songbirds. Species likeability increased for people who fed birds regularly and who could name the species. We found a strong correlation between the number of species that a person could correctly identify and how connected to nature they felt when they watched garden birds. Species richness was preferred over a greater number of individuals of the same species. Although we do not show causation this study suggests that it is possible to increase the well-being benefits that people gain from watching birds at their feeders. This could be done first through a human to bird approach by encouraging regular interactions between people and their garden birds, such as through learning the species names and providing food. Second, it could be achieved through a bird to human approach by increasing garden songbird diversity because the pleasure that a person receives from watching an individual bird at a feeder is dependent not only on its species but also on the diversity of birds at the feeder.

Likeability of Garden Birds: Importance of Species Knowledge & Richness in Connecting People to Nature

PubMed Central

Cox, Daniel T. C.; Gaston, Kevin J.

2015-01-01

Interacting with nature is widely recognised as providing many health and well-being benefits. As people live increasingly urbanised lifestyles, the provision of food for garden birds may create a vital link for connecting people to nature and enabling them to access these benefits. However, it is not clear which factors determine the pleasure that people receive from watching birds at their feeders. These may be dependent on the species that are present, the abundance of individuals and the species richness of birds around the feeders. We quantitatively surveyed urban households from towns in southern England to determine the factors that influence the likeability of 14 common garden bird species, and to assess whether people prefer to see a greater abundance of individuals or increased species richness at their feeders. There was substantial variation in likeability across species, with songbirds being preferred over non-songbirds. Species likeability increased for people who fed birds regularly and who could name the species. We found a strong correlation between the number of species that a person could correctly identify and how connected to nature they felt when they watched garden birds. Species richness was preferred over a greater number of individuals of the same species. Although we do not show causation this study suggests that it is possible to increase the well-being benefits that people gain from watching birds at their feeders. This could be done first through a human to bird approach by encouraging regular interactions between people and their garden birds, such as through learning the species names and providing food. Second, it could be achieved through a bird to human approach by increasing garden songbird diversity because the pleasure that a person receives from watching an individual bird at a feeder is dependent not only on its species but also on the diversity of birds at the feeder. PMID:26560968

Ionoregulatory changes during metamorphosis and salinity exposure of juvenile sea lamprey (Petromyzon marinus L.)

USGS Publications Warehouse

Reis-Santos, P.; McCormick, S.D.; Wilson, J.M.

2008-01-01

Ammocoetes of the anadromous sea lamprey Petromyzon marinus L. spend many years in freshwater before metamorphosing and migrating to sea. Metamorphosis involves the radical transformation from a substrate-dwelling, filter feeder into a free-swimming, parasitic feeder. In the present work we examined osmoregulatory differences between ammocoetes and transformers (metamorphic juveniles), and the effects of salinity acclimation. We measured the expression of key ion-transporting proteins [Na+/K+-ATPase, vacuolar (V)-type H+-ATPase and carbonic anhydrase (CA)] as well as a number of relevant blood parameters (hematocrit, [Na+] and [Cl -]). In addition, immunofluorescence microscopy was used to identify and characterize the distributions of Na+/K+-ATPase, V-type H+-ATPase and CA immunoreactive cells in the gill. Ammocoetes did not survive in the experiments with salinities greater than 10???, whereas survival in high salinity (???25-35???) increased with increased degree of metamorphosis in transformers. Plasma [Na+] and [Cl -] of ammocoetes in freshwater was lower than transformers and increased markedly at 10???. In transformers, plasma ions increased only at high salinity (>25???). Branchial Na+/K+-ATPase levels were ??? tenfold higher in transformers compared to ammocoetes and salinity did not affect expression in either group. However, branchial H +-ATPase expression showed a negative correlation with salinity in both groups. Na+/K+-ATPase immunoreactivity was strongest in transformers and associated with clusters of cells in the interlamellar spaces. H+-ATPase (B subunit) immunoreactivity was localized to epithelial cells not expressing high Na+/K+-ATPase immunoreactivity and having a similar tissue distribution as carbonic anhydrase. The results indicate that branchial Na+/K+-ATPase and salinity tolerance increase in metamorphosing lampreys, and that branchial H+-ATPase is downregulated by salinity.

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

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

Yamano, Noriko; Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp; Watanabe-Kushima, Shoko

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were culturedmore » on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.« less

LOADING REEL AND PERFECTO STRIP STOCK FEEDER FOR #84 WATERBURYFARREL ...

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

LOADING REEL AND PERFECTO STRIP STOCK FEEDER FOR #84 WATERBURY-FARREL (U.S. GOVERNMENT) PRESS. THIS CONTINUOUS-FEED, 2-DRAW, 100 TON PRESS IS ONE OF TWO IN THE U.S. UNDER CONTRACT WITH THE DEPARTMENT OF DEFENSE FOR PRODUCTION OF BULLET JACKETS AND CARTRIDGE CASINGS. - American Brass Foundry, 70 Sayre Street, Buffalo, Erie County, NY

Feeding the Elite: The Evolution of Elite Pathways from Star High Schools to Elite Universities

ERIC Educational Resources Information Center

LeTendre, Gerald K.; Gonzalez, Roger Geertz; Nomi, Takako

2006-01-01

During the last 50 years, private "feeder" schools in Japan came to dominate entry into elite colleges. Intense organizational competition shaped the organizational environment and changed the pathways available to social elites. Compared to Japan, elite private feeders in the US have failed to dominate pathways into elite colleges. In…

ADJUSTABLE OUTPUT RATE CHEMICAL FEEDING EQUIPMENT FOR SWIMMING POOLS. NATIONAL SANITATION FOUNDATION STANDARD NUMBER 19.

ERIC Educational Resources Information Center

National Sanitation Foundation, Ann Arbor, MI.

THE SCOPE OF THIS STANDARD COVERS ADJUSTABLE OUTPUT RATE CHEMICAL FEEDERS, WHETHER USED FOR SOLUTIONS, SLURRIES OR SOLIDS. IT ALSO INCLUDES AUXILIARY EQUIPMENT SUCH AS PUMPS, STRAINERS, TUBING CONNECTIONS, TANKS, INJECTION FITTINGS AND OTHER REQUIRED COMPONENTS. THE FEEDERS DESCRIBED ARE INTENDED TO BE DESIGNED AND USED SPECIFICALLY FOR CHEMICAL…

Norm-Optimal ILC Applied to a High-Speed Rack Feeder

NASA Astrophysics Data System (ADS)

Schindele, Dominik; Aschemann, Harald; Ritzke, Jöran

2010-09-01

Rack feeders as automated conveying systems for high bay rackings are of high practical importance. To shorten the transport times by using trajectories with increased kinematic values accompanying control measures for a reduction of the excited structural vibrations are necessary. In this contribution, the model-based design of a norm-optimal iterative learning control structure is presented. The rack feeder is modelled as an elastic multibody system. For the mathematical description of the bending deflections a Ritz ansatz is introduced. The tracking control design is performed separately for both axes using decentralised state space representations. Both the achievable performance and the resulting tracking accuracy of the proposed control concept are shown by measurement results from the experimental set-up.

Voltage Impacts of Utility-Scale Distributed Wind

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

Allen, A.

2014-09-01

Although most utility-scale wind turbines in the United States are added at the transmission level in large wind power plants, distributed wind power offers an alternative that could increase the overall wind power penetration without the need for additional transmission. This report examines the distribution feeder-level voltage issues that can arise when adding utility-scale wind turbines to the distribution system. Four of the Pacific Northwest National Laboratory taxonomy feeders were examined in detail to study the voltage issues associated with adding wind turbines at different distances from the sub-station. General rules relating feeder resistance up to the point of turbinemore » interconnection to the expected maximum voltage change levels were developed. Additional analysis examined line and transformer overvoltage conditions.« less

Cellular responses to disruption of the permeability barrier in a three-dimensional organotypic epidermal model

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

Ajani, Gati; Sato, Nobuyuki; Mack, Judith A.

2007-08-15

Repeated injury to the stratum corneum of mammalian skin (caused by friction, soaps, or organic solvents) elicits hyperkeratosis and epidermal thickening. Functionally, these changes serve to restore the cutaneous barrier and protect the organism. To better understand the molecular and cellular basis of this response, we have engineered an in vitro model of acetone-induced injury using organotypic epidermal cultures. Rat epidermal keratinocytes (REKs), grown on a collagen raft in the absence of any feeder fibroblasts, developed all the hallmarks of a true epidermis including a well-formed cornified layer. To induce barrier injury, REK cultures were treated with intermittent 30-s exposuresmore » to acetone then were fixed and paraffin-sectioned. After two exposures, increased proliferation (Ki67 and BrdU staining) was observed in basal and suprabasal layers. After three exposures, proliferation became confined to localized buds in the basal layer and increased terminal differentiation was observed (compact hyperkeratosis of the stratum corneum, elevated levels of K10 and filaggrin, and heightened transglutaminase activity). Thus, barrier disruption causes epidermal hyperplasia and/or enhances differentiation, depending upon the extent and duration of injury. Given that no fibroblasts are present in the model, the ability to mount a hyperplastic response to barrier injury is an inherent property of keratinocytes.« less

Considerations for an Earth Relay Satellite with RF and Optical Trunklines

NASA Technical Reports Server (NTRS)

Israel, David J.

2016-01-01

Support for user platforms through the use of optical links to geosynchronous relay spacecraft are expected to be part of the future space communications architecture. The European Data Relay Satellite System (EDRS) has its first node, EDRS-A, in orbit. The EDRS architecture includes space-to-space optical links with a Ka-Band feeder link or trunkline. NASA's Laser Communications Relay Demonstration (LCRD) mission, originally baselined to support a space-to-space optical link relayed with an optical trunkline, has added an Radio Frequency (RF) trunkline. The use of an RF trunkline avoids the outages suffered by an optical trunkline due to clouds, but an RF trunkline will be bandwidth limited. A space relay architecture with both RF and optical trunklines could relay critical realtime data, while also providing a high data volume capacity. This paper considers the relay user scenarios that could be supported, and the implications to the space relay system and operations. System trades such as the amount of onboard processing and storage required, the use of link layer switching vs. network layer routing, and the use of Delay/Disruption Tolerant Networking (DTN) are discussed.

Patterns of relative magnitudes of soil energy channels and their relationships with environmental factors in different ecosystems in Romania.

PubMed

Ciobanu, Marcel; Popovici, Iuliana; Zhao, Jie; Stoica, Ilie-Adrian

2015-12-01

The percentage compositions of soil herbivorous, bacterivorous and fungivorous nematodes in forests, grasslands and scrubs in Romania was analysed. Percentages of nematode abundance, biomass and metabolic footprint methods were used to evaluate the patterns and relative size of herbivory, bacterial- and fungal-mediated channels in organic and mineral soil horizons. Patterns and magnitudes of herbivore, bacterivore and fungivore energy pathways differed for a given ecosystem type and soil depth according to the method used. The relevance of herbivore energy channel increased with soil depth due to higher contribution of root-feeders. Ectoparasites, sedentary parasites and epidermal cell and root hair feeders were the most important contributors to the total biomass and metabolic footprints of herbivores. Metabolic footprint method revealed the general dominance of bacterial-based energy channel in all five types of ecosystems. The influence of altitude and climatic factors on percentages of abundance, biomass and metabolic footprints of herbivores, bacterivores and fungivores decreased with soil depth, whereas the influence of humus content, cation-exchange capacity and base saturation increased. Vegetation, altitude, climate and soil physico-chemical characteristics are important factors that influenced the abundance, biomass and metabolic footprints of herbivores, bacterivores and fungivores.

Patterns of relative magnitudes of soil energy channels and their relationships with environmental factors in different ecosystems in Romania

PubMed Central

Ciobanu, Marcel; Popovici, Iuliana; Zhao, Jie; Stoica, Ilie-Adrian

2015-01-01

The percentage compositions of soil herbivorous, bacterivorous and fungivorous nematodes in forests, grasslands and scrubs in Romania was analysed. Percentages of nematode abundance, biomass and metabolic footprint methods were used to evaluate the patterns and relative size of herbivory, bacterial- and fungal-mediated channels in organic and mineral soil horizons. Patterns and magnitudes of herbivore, bacterivore and fungivore energy pathways differed for a given ecosystem type and soil depth according to the method used. The relevance of herbivore energy channel increased with soil depth due to higher contribution of root-feeders. Ectoparasites, sedentary parasites and epidermal cell and root hair feeders were the most important contributors to the total biomass and metabolic footprints of herbivores. Metabolic footprint method revealed the general dominance of bacterial-based energy channel in all five types of ecosystems. The influence of altitude and climatic factors on percentages of abundance, biomass and metabolic footprints of herbivores, bacterivores and fungivores decreased with soil depth, whereas the influence of humus content, cation-exchange capacity and base saturation increased. Vegetation, altitude, climate and soil physico-chemical characteristics are important factors that influenced the abundance, biomass and metabolic footprints of herbivores, bacterivores and fungivores. PMID:26620189