Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice.

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Fox, Donald A; Hamilton, W Ryan; Johnson, Jerry E; Xiao, Weimin; Chaney, Shawntay; Mukherjee, Shradha; Miller, Diane B; O'Callaghan, James P

2011-11-01

Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ~25 and ~40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. Copyright © 2011 Elsevier Inc. All rights reserved.

TOPOGRAPHIC ORGANIZATION AND SPECIALIZED AREAS IN THE RETINA OF Callopistes palluma: GANGLION CELL LAYER

OpenAIRE

Inzunza, Oscar; Barros B., Zitta; Bravo, Hermes

1998-01-01

In this paper we analyze the topographic distribution and cell body size of neurons (ganglion and displaced amacrine) of layer 8 of the retina in the chilean reptile Callopistes palluma; using whole mount retinaswith nissl stain. Callopistes palluma retina has an area centralis without fovea in which the ganglion cell density amounts 20.000 cells / µm2 while the displaced amacrine neurons is about 7.000 cells / µm2. This neural density decreased gradually towards the peripheral retina. A hor...

ON Cone Bipolar Cell Axonal Synapses in the OFF Inner Plexiform Layer of the Rabbit Retina

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Lauritzen, J. Scott; Anderson, James R.; Jones, Bryan W.; Watt, Carl B.; Mohammed, Shoeb; Hoang, John V.; Marc, Robert E.

2012-01-01

Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make non-canonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscope (ATEM) imaging, molecular tagging, tracing, and rendering of ≈ 400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include GABA-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs) and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON-OFF amacrine cells. Many of these ON-OFF GACs target ON cone bipolar cell axons, ON γACs and/or ON-OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON-OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells. PMID:23042441

Retinal Ganglion Cell Distribution and Spatial Resolving Power in Deep-Sea Lanternfishes (Myctophidae)

KAUST Repository

De Busserolles, Fanny

2014-01-01

Topographic analyses of retinal ganglion cell density are very useful in providing information about the visual ecology of a species by identifying areas of acute vision within the visual field (i.e. areas of high cell density). In this study, we investigated the neural cell distribution in the ganglion cell layer of a range of lanternfish species belonging to 10 genera. Analyses were performed on wholemounted retinas using stereology. Topographic maps were constructed of the distribution of all neurons and both ganglion and amacrine cell populations in 5 different species from Nissl-stained retinas using cytological criteria. Amacrine cell distribution was also examined immunohistochemically in 2 of the 5 species using anti-parvalbumin antibody. The distributions of both the total neuron and the amacrine cell populations were aligned in all of the species examined, showing a general increase in cell density toward the retinal periphery. However, when the ganglion cell population was topographically isolated from the amacrine cell population, which comprised up to 80% of the total neurons within the ganglion cell layer, a different distribution was revealed. Topographic maps of the true ganglion cell distribution in 18 species of lanternfishes revealed well-defined specializations in different regions of the retina. Different species possessed distinct areas of high ganglion cell density with respect to both peak density and the location and/or shape of the specialized acute zone (i.e. elongated areae ventro-temporales, areae temporales and large areae centrales). The spatial resolving power was calculated to be relatively low (varying from 1.6 to 4.4 cycles per degree), indicating that myctophids may constitute one of the less visually acute groups of deep-sea teleosts. The diversity in retinal specializations and spatial resolving power within the family is assessed in terms of possible ecological functions and evolutionary history.

Asymmetry between ON and OFF α ganglion cells of mouse retina: integration of signal and noise from synaptic inputs.

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Freed, Michael A

2017-11-15

Bipolar and amacrine cells presynaptic to the ON sustained α cell of mouse retina provide currents with a higher signal-to-noise power ratio (SNR) than those presynaptic to the OFF sustained α cell. Yet the ON cell loses proportionately more SNR from synaptic inputs to spike output than the OFF cell does. The higher SNR of ON bipolar cells at the beginning of the ON pathway compensates for losses incurred by the ON ganglion cell, and improves the processing of positive contrasts. ON and OFF pathways in the retina include functional pairs of neurons that, at first glance, appear to have symmetrically similar responses to brightening and darkening, respectively. Upon careful examination, however, functional pairs exhibit asymmetries in receptive field size and response kinetics. Until now, descriptions of how light-adapted retinal circuitry maintains a preponderance of signal over the noise have not distinguished between ON and OFF pathways. Here I present evidence of marked asymmetries between members of a functional pair of sustained α ganglion cells in the mouse retina. The ON cell exhibited a proportionately greater loss of signal-to-noise power ratio (SNR) from its presynaptic arrays to its postsynaptic currents. Thus the ON cell combines signal and noise from its presynaptic arrays of bipolar and amacrine cells less efficiently than the OFF cell does. Yet the inefficiency of the ON cell is compensated by its presynaptic arrays providing a higher SNR than the arrays presynaptic to the OFF cell, apparently to improve visual processing of positive contrasts. Dynamic clamp experiments were performed that introduced synaptic conductances into ON and OFF cells. When the amacrine-modulated conductance was removed, the ON cell's spike train exhibited an increase in SNR. The OFF cell, however, showed the opposite effect of removing amacrine input, which was a decrease in SNR. Thus ON and OFF cells have different modes of synaptic integration with direct effects on

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

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Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

2015-10-29

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. Published by Elsevier

Thyroid Hormone Signaling in the Mouse Retina.

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Patrick Arbogast

Full Text Available Thyroid hormone is a crucial regulator of gene expression in the developing and adult retina. Here we sought to map sites of thyroid hormone signaling at the cellular level using the transgenic FINDT3 reporter mouse model in which neurons express β-galactosidase (β-gal under the control of a hybrid Gal4-TRα receptor when triiodothyronine (T3 and cofactors of thyroid receptor signaling are present. In the adult retina, nearly all neurons of the ganglion cell layer (GCL, ganglion cells and displaced amacrine cells showed strong β-gal labeling. In the inner nuclear layer (INL, a minority of glycineric and GABAergic amacrine cells showed β-gal labeling, whereas the majority of amacrine cells were unlabeled. At the level of amacrine types, β-gal labeling was found in a large proportion of the glycinergic AII amacrines, but only in a small proportion of the cholinergic/GABAergic 'starburst' amacrines. At postnatal day 10, there also was a high density of strongly β-gal-labeled neurons in the GCL, but only few amacrine cells were labeled in the INL. There was no labeling of bipolar cells, horizontal cells and Müller glia cells at both stages. Most surprisingly, the photoreceptor somata in the outer nuclear layer also showed no β-gal label, although thyroid hormone is known to control cone opsin expression. This is the first record of thyroid hormone signaling in the inner retina of an adult mammal. We hypothesize that T3 levels in photoreceptors are below the detection threshold of the reporter system. The topographical distribution of β-gal-positive cells in the GCL follows the overall neuron distribution in that layer, with more T3-signaling cells in the ventral than the dorsal half-retina.

Localization of glycine-containing neurons in the Macaca monkey retina

International Nuclear Information System (INIS)

Hendrickson, A.E.; Koontz, M.A.; Pourcho, R.G.; Sarthy, P.V.; Goebel, D.J.

1988-01-01

Autoradiography following 3H-glycine (Gly) uptake and immunocytochemistry with a Gly-specific antiserum were used to identify neurons in Macaca monkey retina that contain a high level of this neurotransmitter. High-affinity uptake of Gly was shown to be sodium dependent whereas release of both endogenous and accumulated Gly was calcium dependent. Neurons labeling for Gly included 40-46% of the amacrine cells and nearly 40% of the bipolars. Synaptic labeling was seen throughout the inner plexiform layer (IPL) but with a preferential distribution in the inner half. Bands of labeled puncta occurred in S2, S4, and S5. Both light and postembedding electron microscopic (EM) immunocytochemistry identified different types of amacrine and bipolar cell bodies and their synaptic terminals. The most heavily labeled Gly+ cell bodies typically were amacrine cells having a single, thick, basal dendrite extending deep into the IPL and, at the EM level, electron-dense cytoplasm and prominent nuclear infoldings. This cell type may be homologous with the Gly2 cell in human retina and the AII/Gly2 of cat retina. Gly+ amacrines synapse most frequently onto Gly- amacrines and both Gly- and Gly+ bipolars. Gly+ bipolar cells appeared to be cone bipolars because their labeled dendrites could be traced only to cone pedicles. The pattern of these labeled dendritic trees indicated that both diffuse and midget types of biopolars were Gly+. The EM distribution of labeled synapses showed Gly+ amacrine synapses throughout the IPL, but these composed only 11-23% of the amacrine population. Most of the Gly+ bipolar terminals were in the inner IPL, where 70% of all bipolar terminals were labeled

Cell-type specific roles for PTEN in establishing a functional retinal architecture.

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Robert Cantrup

Full Text Available The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and

Localization, distribution, and connectivity of neuropeptide Y in the human and porcine retinas-A comparative study.

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Christiansen, Anders Tolstrup; Kiilgaard, Jens Folke; Klemp, Kristian; Woldbye, David Paul Drucker; Hannibal, Jens

2018-04-17

Neuropeptide Y (NPY) is a peptide neurotransmitter abundantly expressed in the mammalian retina. Since its discovery, NPY has been studied in retinas of several species, but detailed characterization of morphology, cell-type, and connectivity has never been conducted in larger mammals including humans and pigs. As the pig due to size and cellular composition is a well-suited animal for retinal research, we chose to compare the endogenous NPY system of the human retina to that of pigs to support future research in this field. In the present study, using immunohistochemistry, confocal microscopy and 3D reconstructions, we found NPY to be expressed in GABAergic and calretinin-immunoreactive (-ir) amacrine cells of both species as well as parvalbumin-ir amacrine cells of humans. Furthermore, we identified at least two different types of medium- to wide-field NPY-ir amacrine cells. Finally, we detected likely synaptic appositions between the NPY-ir amacrine cells and melanopsin- and nonmelanopsin-ir ganglion cells, GABAergic and dopaminergic amacrine cells, rod bipolar cells, and horizontal cells, suggesting that NPY-ir cells play diverse roles in modulation of both image and non-image forming retinal signaling. These findings extend existing knowledge on NPY and NPY-expressing cells in the human and porcine retina showing a high degree of comparability. The extensive distribution and connectivity of NPY-ir cells described in the present study further highlights the potential importance of NPY signaling in retinal function. © 2018 Wiley Periodicals, Inc.

Glycinergic pathways in the goldfish retina

International Nuclear Information System (INIS)

Marc, R.E.; Lam, D.M.

1981-01-01

Autoradiographic localization of high affinity [3H]glycine uptake in the retina of the goldfish has been used to study some anatomical and physiological properties of potentially glycinergic neurons. There are two classes of retinal cells exhibiting high affinity glycine uptake: Aa amacrine cells and I2 interplexiform cells. Aa amacrine cells constitute about 20% of the somas in the amacrine cell layer and send their dendrites to the middle of the inner plexiform layer. There they are both pre- and postsynaptic primarily to other amacrine cells. Photic modulation of glycine uptake indicates that they are probably red-hyperpolarizing/green-depolarizing neurons. I2 interplexiform cells are a newly discovered type of interplexiform cell; in the outer plexiform layer, they receive direct synaptic input from the somas of red-dominated GABAergic H1 horizontal cells and are apparently presynaptic to dendrites of unidentified types of horizontal cells. The connections of I2 interplexiform cells have not been successfully characterized in the inner plexiform layer. These findings extend our knowledge of neurochemically specific pathways in the cyprinid retina and indicate that glycine, like GABA, is a neurotransmitter primarily involved with circuits coding ''red'' information

From retinal waves to activity-dependent retinogeniculate map development.

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Markowitz, Jeffrey; Cao, Yongqiang; Grossberg, Stephen

2012-01-01

A neural model is described of how spontaneous retinal waves are formed in infant mammals, and how these waves organize activity-dependent development of a topographic map in the lateral geniculate nucleus, with connections from each eye segregated into separate anatomical layers. The model simulates the spontaneous behavior of starburst amacrine cells and retinal ganglion cells during the production of retinal waves during the first few weeks of mammalian postnatal development. It proposes how excitatory and inhibitory mechanisms within individual cells, such as Ca(2+)-activated K(+) channels, and cAMP currents and signaling cascades, can modulate the spatiotemporal dynamics of waves, notably by controlling the after-hyperpolarization currents of starburst amacrine cells. Given the critical role of the geniculate map in the development of visual cortex, these results provide a foundation for analyzing the temporal dynamics whereby the visual cortex itself develops.

From retinal waves to activity-dependent retinogeniculate map development.

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Jeffrey Markowitz

Full Text Available A neural model is described of how spontaneous retinal waves are formed in infant mammals, and how these waves organize activity-dependent development of a topographic map in the lateral geniculate nucleus, with connections from each eye segregated into separate anatomical layers. The model simulates the spontaneous behavior of starburst amacrine cells and retinal ganglion cells during the production of retinal waves during the first few weeks of mammalian postnatal development. It proposes how excitatory and inhibitory mechanisms within individual cells, such as Ca(2+-activated K(+ channels, and cAMP currents and signaling cascades, can modulate the spatiotemporal dynamics of waves, notably by controlling the after-hyperpolarization currents of starburst amacrine cells. Given the critical role of the geniculate map in the development of visual cortex, these results provide a foundation for analyzing the temporal dynamics whereby the visual cortex itself develops.

Meis1 regulates Foxn4 expression during retinal progenitor cell differentiation

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Mohammed M. Islam

2013-09-01

The transcription factor forkhead box N4 (Foxn4 is a key regulator in a variety of biological processes during development. In particular, Foxn4 plays an essential role in the genesis of horizontal and amacrine neurons from neural progenitors in the vertebrate retina. Although the functions of Foxn4 have been well established, the transcriptional regulation of Foxn4 expression during progenitor cell differentiation remains unclear. Here, we report that an evolutionarily conserved 129 bp noncoding DNA fragment (Foxn4CR4.2 or CR4.2, located ∼26 kb upstream of Foxn4 transcription start site, functions as a cis-element for Foxn4 regulation. CR4.2 directs gene expression in Foxn4-positive cells, primarily in progenitors, differentiating horizontal and amacrine cells. We further determined that the gene regulatory activity of CR4.2 is modulated by Meis1 binding motif, which is bound and activated by Meis1 transcription factor. Deletion of the Meis1 binding motif or knockdown of Meis1 expression abolishes the gene regulatory activity of CR4.2. In addition, knockdown of Meis1 expression diminishes the endogenous Foxn4 expression and affects cell lineage development. Together, we demonstrate that CR4.2 and its interacting Meis1 transcription factor play important roles in regulating Foxn4 expression during early retinogenesis. These findings provide new insights into molecular mechanisms that govern gene regulation in retinal progenitors and specific cell lineage development.

The long noncoding RNA RNCR2 directs mouse retinal cell specification

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Blackshaw Seth

2010-05-01

Full Text Available Abstract Background Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation. Results We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation. Conclusion These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.

Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

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Chintala, Shravan; Cheng, Mei; Zhang, Xiao

2015-01-01

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and

The Transient Intermediate Plexiform Layer, a Plexiform Layer-like Structure Temporarily Existing in the Inner Nuclear Layer in Developing Rat Retina.

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Park, Hyung Wook; Kim, Hong-Lim; Park, Yong Soo; Kim, In-Beom

2018-02-01

The retina is a highly specialised part of the brain responsible for visual processing. It is well-laminated; three layers containing five different types of neurons are compartmentalised by two synaptic layers. Among the retinal layers, the inner nuclear layer (INL) is composed of horizontal, bipolar, and amacrine cell types. Bipolar cells form one sublayer in the distal half of the IPL, while amacrine cells form another sublayer in the proximal half, without any border-like structure. Here, we report that a plexiform layer-like structure exists temporarily in the border between the bipolar and amacrine sublayers in the INL in the rat retina during retinal development. This transient intermediate plexiform layer (TIPL) appeared at postnatal day (PD) 7 and then disappeared around PD 12. Most apoptotic cells in the INL were found near the TIPL. These results suggest that the TIPL may contribute to the formation of sublayers and the cell number limit in the INL.

Autoradiographic analysis of 3H-glutamate, 3H-dopamine, and 3H-GABA accumulation in rabbit retina after kainic acid treatment

International Nuclear Information System (INIS)

Hampton, C.K.; Redburn, D.A.

1983-01-01

We have previously reported that exposure of isolated rabbit retina to 10(-3) M kainic acid produces profound morphological changes in specific retinal neurons (Hampton et al, 1981). We noted specific swelling of horizontal cell bodies and neurites, necrosis of cell bodies in the amacrine and ganglion cell layers, and swelling of elements in the inner plexiform layer. We now report a differential sensitivity to kainic acid of specific subclasses of amacrine cells autoradiographically labeled with 3H-glutamate, 3H-GABA, or 3H-dopamine. Three different effects were observed: (1) Labeling of neurons after incubation in 3H-glutamate was uniformly reduced while labeling of glia was much less affected. (2) The accumulation of 3H-dopamine was also decreased by kainic acid in two of the three labeled bands of the inner plexiform layer. The outermost labeled band was insensitive to kainic acid at the highest concentration tested (10(-2) M). These findings provide a basis for the subclassification of dopaminergic amacrine cells into at least two subclasses based on their sensitivity to kainic acid. (3) Kainic acid caused a dramatic increase in the labeling of GABAergic amacrine cell bodies and their terminals. This increased intensity may reflect a compensatory increase in uptake activity in response to kainic acid-induced depletion of endogenous GABA stores. These results confirm the highly toxic nature of kainic acid and demonstrate a high degree of specificity and complexity in its action in the retina

Melanopsin expressing human retinal ganglion cells: Subtypes, distribution, and intraretinal connectivity.

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Hannibal, Jens; Christiansen, Anders Tolstrup; Heegaard, Steffen; Fahrenkrug, Jan; Kiilgaard, Jens Folke

2017-06-01

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N- and C-terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1 (GDM1)." Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin-ir (0.63-0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin-ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin-ir RGCs, which most likely are involved in different functions. © 2017 Wiley Periodicals, Inc.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells.

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Walker, Marquis T; Rupp, Alan; Elsaesser, Rebecca; Güler, Ali D; Sheng, Wenlong; Weng, Shijun; Berson, David M; Hattar, Samer; Montell, Craig

2015-10-15

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2(-/-) mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2(-/-) were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2(-/-) mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light. © 2015 Walker et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Introduction to solar cell production

International Nuclear Information System (INIS)

Kim, Gyeong Hae; Lee, Jun Sin

2009-08-01

This book introduces solar cell production. It is made up eight chapters, which are summary of solar cell with structure and prospect of the business, special variable of solar cell on light of the sun and factor causing variable of solar cell, production of solar cell with surface texturing, diffusion, metal printing dry and firing and edge isolation, process of solar cell on silicone wafer for solar cell, forming of electrodes, introduction of thin film solar cell on operating of solar cell, process of production and high efficiency of thin film solar cell, sorting of solar cell and production with background of silicone solar cell and thin film solar cell, structure and production of thin film solar cell and compound solar cell, introduction of solar cell module and the Industrial condition and prospect of solar cell.

Strategies for fuel cell product development. Developing fuel cell products in the technology supply chain

International Nuclear Information System (INIS)

Hellman, H.L.

2004-01-01

Due to the high cost of research and development and the broad spectrum of knowledge and competences required to develop fuel cell products, many product-developing firms outsource fuel cell technology, either partly or completely. This article addresses the inter-firm process of fuel cell product development from an Industrial Design Engineering perspective. The fuel cell product development can currently be characterised by a high degree of economic and technical uncertainty. Regarding the technology uncertainty: product-developing firms are more often then not unfamiliar with fuel cell technology technology. Yet there is a high interface complexity between the technology supplied and the product in which it is to be incorporated. In this paper the information exchange in three current fuel cell product development projects is analysed to determine the information required by a product designer to develop a fuel cell product. Technology transfer literature suggests that transfer effectiveness is greatest when the type of technology (technology uncertainty) and the type of relationship between the technology supplier and the recipient are carefully matched. In this line of thinking this paper proposes that the information required by a designer, determined by the design strategy and product/system volume, should be met by an appropriate level of communication interactivity with a technology specialist. (author)

Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

Science.gov (United States)

Eldred, Megan K; Charlton-Perkins, Mark; Muresan, Leila; Harris, William A

2017-03-15

To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process. © 2017. Published by The Company of Biologists Ltd.

Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells.

Science.gov (United States)

Elgueta, Claudio; Leroy, Felix; Vielma, Alex H; Schmachtenberg, Oliver; Palacios, Adrian G

2018-02-15

A17 amacrine cells are an important part of the scotopic pathway. Their synaptic varicosities receive glutamatergic inputs from rod bipolar cells (RBC) and release GABA onto the same RBC terminal, forming a reciprocal feedback that shapes RBC depolarization. Here, using patch-clamp recordings, we characterized electrical coupling between A17 cells of the rat retina and report the presence of strongly interconnected and non-coupled A17 cells. In coupled A17 cells, evoked currents preferentially flow out of the cell through GJs and cross-synchronization of presynaptic signals in a pair of A17 cells is correlated to their coupling degree. Moreover, we demonstrate that stimulation of one A17 cell can induce electrical and calcium transients in neighboring A17 cells, thus confirming a functional flow of information through electrical synapses in the A17 coupled network. Finally, blocking GJs caused a strong decrease in the amplitude of the inhibitory feedback onto RBCs. We therefore propose that electrical coupling between A17 cells enhances feedback onto RBCs by synchronizing and facilitating GABA release from inhibitory varicosities surrounding each RBC axon terminal. GJs between A17 cells are therefore critical in shaping the visual flow through the scotopic pathway.

Light adaptation alters the source of inhibition to the mouse retinal OFF pathway

Science.gov (United States)

Mazade, Reece E.

2013-01-01

Sensory systems must avoid saturation to encode a wide range of stimulus intensities. One way the retina accomplishes this is by using both dim-light-sensing rod and bright-light-sensing cone photoreceptor circuits. OFF cone bipolar cells are a key point in this process, as they receive both excitatory input from cones and inhibitory input from AII amacrine cells via the rod pathway. However, in addition to AII amacrine cell input, other inhibitory inputs from cone pathways also modulate OFF cone bipolar cell light signals. It is unknown how these inhibitory inputs to OFF cone bipolar cells change when switching between rod and cone pathways or whether all OFF cone bipolar cells receive rod pathway input. We found that one group of OFF cone bipolar cells (types 1, 2, and 4) receive rod-mediated inhibitory inputs that likely come from the rod-AII amacrine cell pathway, while another group of OFF cone bipolar cells (type 3) do not. In both cases, dark-adapted rod-dominant light responses showed a significant contribution of glycinergic inhibition, which decreased with light adaptation and was, surprisingly, compensated by an increase in GABAergic inhibition. As GABAergic input has distinct timing and spatial spread from glycinergic input, a shift from glycinergic to GABAergic inhibition could significantly alter OFF cone bipolar cell signaling to downstream OFF ganglion cells. Larger GABAergic input could reflect an adjustment of OFF bipolar cell spatial inhibition, which may be one mechanism that contributes to retinal spatial sensitivity in the light. PMID:23926034

Abnormal Glycogen Storage by Retinal Neurons in Diabetes.

Science.gov (United States)

Gardiner, Tom A; Canning, Paul; Tipping, Nuala; Archer, Desmond B; Stitt, Alan W

2015-12-01

It is widely held that neurons of the central nervous system do not store glycogen and that accumulation of the polysaccharide may cause neurodegeneration. Since primary neural injury occurs in diabetic retinopathy, we examined neuronal glycogen status in the retina of streptozotocin-induced diabetic and control rats. Glycogen was localized in eyes of streptozotocin-induced diabetic and control rats using light microscopic histochemistry and electron microscopy, and correlated with immunohistochemical staining for glycogen phosphorylase and phosphorylated glycogen synthase (pGS). Electron microscopy of 2-month-old diabetic rats (n = 6) showed massive accumulations of glycogen in the perinuclear cytoplasm of many amacrine neurons. In 4-month-old diabetic rats (n = 11), quantification of glycogen-engorged amacrine cells showed a mean of 26 cells/mm of central retina (SD ± 5), compared to 0.5 (SD ± 0.2) in controls (n = 8). Immunohistochemical staining for glycogen phosphorylase revealed strong expression in amacrine and ganglion cells of control retina, and increased staining in cell processes of the inner plexiform layer in diabetic retina. In control retina, the inactive pGS was consistently sequestered within the cell nuclei of all retinal neurons and the retinal pigment epithelium (RPE), but in diabetics nuclear pGS was reduced or lost in all classes of retinal cell except the ganglion cells and cone photoreceptors. The present study identifies a large population of retinal neurons that normally utilize glycogen metabolism but show pathologic storage of the polysaccharide during uncontrolled diabetes.

Tensor Product of Polygonal Cell Complexes

OpenAIRE

Chien, Yu-Yen

2017-01-01

We introduce the tensor product of polygonal cell complexes, which interacts nicely with the tensor product of link graphs of complexes. We also develop the unique factorization property of polygonal cell complexes with respect to the tensor product, and study the symmetries of tensor products of polygonal cell complexes.

Xenopus laevis Retinal Ganglion Cell Dendritic Arbors Develop Independently of Visual Stimulation

Directory of Open Access Journals (Sweden)

Barbara Lom

2004-01-01

Full Text Available Newly formed neurons must locate their appropriate target cells and then form synaptic connections with these targets in order to establish a functional nervous system. In the vertebrate retina, retinal ganglion cell (RGC dendrites extend from the cell body and form synapses with nearby amacrine and bipolar cells. RGC axons, however, exit the retina and synapse with the dendrites of midbrain neurons in the optic tectum. We examined how visual stimulation influenced Xenopus RGC dendritic arborization. Neuronal activity is known to be an important factor in shaping dendritic and axonal arborization. Thus, we reared tadpoles in dark and light environments then used rhodamine dextran retrograde labeling to identify RGCs in the retina. When we compared RGC dendritic arbors from tadpoles reared in dark and light environments, we found no morphological differences, suggesting that physiological visual activity did not contribute to the morphological development of Xenopus RGC dendritic arbors.

Cytogenesis in the monkey retina

International Nuclear Information System (INIS)

La Vail, M.M.; Rapaport, D.H.; Rakic, P.

1991-01-01

Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Mueller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Mueller, and rod cells have a similar time of origin

REVIEW ARTICLE Therapeutic avenues for hereditary forms of ...

Indian Academy of Sciences (India)

chitra

degenerations and dystrophies [Retinal Information Network, RetNet. .... out, and nutrients to diffuse in, while preventing the host immune system from attacking the ... amacrine cells, as well as the retinal nerve fiber layer, that are relatively ...

Cell fusion in tumor progression: the isolation of cell fusion products by physical methods

Directory of Open Access Journals (Sweden)

Vincitorio Massimo

2011-09-01

Full Text Available Abstract Background Cell fusion induced by polyethylene glycol (PEG is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks. Results Here we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia. Conclusions In the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

Solar Cell Production in Nigeria: Prospects, Options and Problems

International Nuclear Information System (INIS)

Fasasi, A. Y.; Siyanbola, W.O.; Ibitoye, F. I.; Pelemo, D. A.

2002-01-01

The prospects and problems facing solar cell production in Nigeria are discussed. The paper reviews many proven solar cell materials in terms of their current efficiencies and production costs. Silicon solar cell production appears to be the best technology option for Nigeria because of the abundant quartz sand and waste products from our phosphate fertiliser company that can be employed as starting materials to produce solar grade silicon. Factors affecting solar cell efficiency, choice of solar cell as well as financial and material problems limiting the progress on silicon solar cell production are also discussed. Finally, the paper recommends the simultaneous production of solar grade silicon and coordinated development of the balance of system components as first steps towards actualizing this objective

Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina.

Science.gov (United States)

Gábriel, Robert; de Souza, Sunita; Ziff, Edward B; Witkovsky, Paul

2002-07-22

We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain. Copyright 2002 Wiley-Liss, Inc.

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

Science.gov (United States)

Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

2015-01-01

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

Low-cost production of solar-cell panels

Science.gov (United States)

Bickler, D. B.; Gallagher, B. D.; Sanchez, L. E.

1980-01-01

Large-scale production model combines most modern manufacturing techniques to produce silicon-solar-cell panels of low costs by 1982. Model proposes facility capable of operating around the clock with annual production capacity of 20 W of solar cell panels.

Bovine mammary stem cells: Cell biology meets production agriculture

Science.gov (United States)

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue ...

Fuel Cell Electric Vehicle Composite Data Products | Hydrogen and Fuel

Science.gov (United States)

Cells | NREL Vehicle Composite Data Products Fuel Cell Electric Vehicle Composite Data Products The following composite data products (CDPs) focus on current fuel cell electric vehicle evaluations Cell Operation Hour Groups CDP FCEV 39, 2/19/16 Comparison of Fuel Cell Stack Operation Hours and Miles

Microbial electrolysis cells as innovative technology for hydrogen production

International Nuclear Information System (INIS)

Chorbadzhiyska, Elitsa; Hristov, Georgi; Mitov, Mario; Hubenova, Yolina

2011-01-01

Hydrogen production is becoming increasingly important in view of using hydrogen in fuel cells. However, most of the production of hydrogen so far comes from the combustion of fossil fuels and water electrolysis. Microbial Electrolysis Cell (MEC), also known as Bioelectrochemically Assisted Microbial Reactor, is an ecologically clean, renewable and innovative technology for hydrogen production. Microbial electrolysis cells produce hydrogen mainly from waste biomass assisted by various bacteria strains. The principle of MECs and their constructional elements are reviewed and discussed. Keywords: microbial Electrolysis Cells, hydrogen production, waste biomass purification

Critical role of NKT cells in posttransplant alloantibody production.

Science.gov (United States)

Zimmerer, J M; Swamy, P; Sanghavi, P B; Wright, C L; Abdel-Rasoul, M; Elzein, S M; Brutkiewicz, R R; Bumgardner, G L

2014-11-01

We previously reported that posttransplant alloantibody production in CD8-deficient hosts is IL-4+ CD4+ T cell-dependent and IgG1 isotype-dominant. The current studies investigated the hypothesis that IL-4-producing natural killer T cells (NKT cells) contribute to maximal alloantibody production. To investigate this, alloantibody levels were examined in CD8-deficient WT, CD1d KO and Jα18 KO transplant recipients. We found that the magnitude of IgG1 alloantibody production was critically dependent on the presence of type I NKT cells, which are activated by day 1 posttransplant. Unexpectedly, type I NKT cell contribution to enhanced IgG1 alloantibody levels was interferon-γ-dependent and IL-4-independent. Cognate interactions between type I NKT and B cells alone do not stimulate alloantibody production. Instead, NKT cells appear to enhance maturation of IL-4+ CD4+ T cells. To our knowledge, this is the first report to substantiate a critical role for type I NKT cells in enhancing in vivo antibody production in response to endogenous antigenic stimuli. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

Effects of irradiation on cytokine production in glioma cell lines

Energy Technology Data Exchange (ETDEWEB)

Yamanaka, Ryuya; Tanaka, Ryuichi; Yoshida, Seiichi [Niigata Univ. (Japan). Brain Research Inst.

1993-11-01

The effects of irradiation on cytokine production in glioma cell lines, NP1, NP2 and NP3, were studied. Culture supernatants were collected after 6, 24, 48 or 72 hours and the concentrations of interleukin (IL)-6 and IL-8 measured by enzyme-linked immunosorbent assay. Spontaneous and IL-1[beta]-stimulated productions were analyzed. Some cells were given a single dose of Lineac irradiation (10 or 20 Gy). Production of IL-6 (with or without IL-1[beta] stimulation) increased gradually to a maximum after 72 hours, more in the 20 Gy-irradiated cells than 10 Gy cells (p<0.01). Production of IL-8 increased gradually to a maximum after 48 or 72 hours. Spontaneous production of IL-8 increased more in 20 Gy-irradiated cells than 10 Gy cells after 6 and 24 hours (p<0.01), but increased more in 10 Gy cells than 20 Gy cells after 48 and 72 hours (p<0.01). The production of IL-8 stimulated by IL-1[beta] increased more in 10 Gy cells than 20 Gy cells 24 hours later (p<0.01). IL-6 and IL-8 production differed in the response to irradiation. Our data suggest that bidirectional communication between the immune system and glioma cells changes after radiotherapy. (author).

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

Science.gov (United States)

D'Amour, Kevin A; Bang, Anne G; Eliazer, Susan; Kelly, Olivia G; Agulnick, Alan D; Smart, Nora G; Moorman, Mark A; Kroon, Evert; Carpenter, Melissa K; Baetge, Emmanuel E

2006-11-01

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Vsx2 in the zebrafish retina: restricted lineages through derepression

Directory of Open Access Journals (Sweden)

Higashijima Shin-ichi

2009-04-01

Full Text Available Abstract Background The neurons in the vertebrate retina arise from multipotent retinal progenitor cells (RPCs. It is not clear, however, which progenitors are multipotent or why they are multipotent. Results In this study we show that the homeodomain transcription factor Vsx2 is initially expressed throughout the retinal epithelium, but later it is downregulated in all but a minor population of bipolar cells and all Müller glia. The Vsx2-negative daughters of Vsx2-positive RPCs divide and give rise to all other cell types in the retina. Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates. Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2. Conclusion Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential.

Bipolar Cell-Photoreceptor Connectivity in the Zebrafish (Danio rerio) Retina

Science.gov (United States)

Li, Yong N.; Tsujimura, Taro; Kawamura, Shoji; Dowling, John E.

2013-01-01

Bipolar cells convey luminance, spatial and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole-mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI-labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly-ordered mosaic: rows of alternating blue- (B) and ultraviolet-sensitive (UV) single cones alternate with rows of red- (R) and green-sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which – G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod and RRod bipolar cells – accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further sub-divided into ON, OFF, and ON-OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the 9 common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels. PMID:22907678

A temperature-controlled photoelectrochemical cell for quantitative product analysis

Science.gov (United States)

Corson, Elizabeth R.; Creel, Erin B.; Kim, Youngsang; Urban, Jeffrey J.; Kostecki, Robert; McCloskey, Bryan D.

2018-05-01

In this study, we describe the design and operation of a temperature-controlled photoelectrochemical cell for analysis of gaseous and liquid products formed at an illuminated working electrode. This cell is specifically designed to quantitatively analyze photoelectrochemical processes that yield multiple gas and liquid products at low current densities and exhibit limiting reactant concentrations that prevent these processes from being studied in traditional single chamber electrolytic cells. The geometry of the cell presented in this paper enables front-illumination of the photoelectrode and maximizes the electrode surface area to electrolyte volume ratio to increase liquid product concentration and hence enhances ex situ spectroscopic sensitivity toward them. Gas is bubbled through the electrolyte in the working electrode chamber during operation to maintain a saturated reactant concentration and to continuously mix the electrolyte. Gaseous products are detected by an in-line gas chromatograph, and liquid products are analyzed ex situ by nuclear magnetic resonance. Cell performance was validated by examining carbon dioxide reduction on a silver foil electrode, showing comparable results both to those reported in the literature and identical experiments performed in a standard parallel-electrode electrochemical cell. To demonstrate a photoelectrochemical application of the cell, CO2 reduction experiments were carried out on a plasmonic nanostructured silver photocathode and showed different product distributions under dark and illuminated conditions.

Methods and practices to diversify cell-based products.

Science.gov (United States)

Vertès, Alain A

2017-12-15

Medicinal signaling cell (MSC)-based products represent emerging treatments in various therapeutic areas including cardiometabolic, inflammation, autoimmunity, orthopedics, wound healing and oncology. Exploring innovation beyond minimally manipulated plastic-adherent ex vivo expanded allogeneic MSCs enables product delineation. Product delineation is on the critical path to maximize clinical benefits and market access. An innovation framework is presented here along various innovation dimensions comprising composition-of-matter by means of positive cell surface markers, formulation varying for example the cell dose or the preservation mode and medium, manufacturing to adapt the secretome of MSCs to the condition of interest, the mode of delivery and corresponding delivery devices, as well as molecular engineering and biomarkers. The rationale of the innovation space thus described applies generally to all cell-based therapies.

Engineering microbial cell factories for the production of plant natural products: from design principles to industrial-scale production.

Science.gov (United States)

Liu, Xiaonan; Ding, Wentao; Jiang, Huifeng

2017-07-19

Plant natural products (PNPs) are widely used as pharmaceuticals, nutraceuticals, seasonings, pigments, etc., with a huge commercial value on the global market. However, most of these PNPs are still being extracted from plants. A resource-conserving and environment-friendly synthesis route for PNPs that utilizes microbial cell factories has attracted increasing attention since the 1940s. However, at the present only a handful of PNPs are being produced by microbial cell factories at an industrial scale, and there are still many challenges in their large-scale application. One of the challenges is that most biosynthetic pathways of PNPs are still unknown, which largely limits the number of candidate PNPs for heterologous microbial production. Another challenge is that the metabolic fluxes toward the target products in microbial hosts are often hindered by poor precursor supply, low catalytic activity of enzymes and obstructed product transport. Consequently, despite intensive studies on the metabolic engineering of microbial hosts, the fermentation costs of most heterologously produced PNPs are still too high for industrial-scale production. In this paper, we review several aspects of PNP production in microbial cell factories, including important design principles and recent progress in pathway mining and metabolic engineering. In addition, implemented cases of industrial-scale production of PNPs in microbial cell factories are also highlighted.

Multiple cone pathways are involved in photic regulation of retinal dopamine.

Science.gov (United States)

Qiao, Sheng-Nan; Zhang, Zhijing; Ribelayga, Christophe P; Zhong, Yong-Mei; Zhang, Dao-Qi

2016-06-30

Dopamine is a key neurotransmitter in the retina and plays a central role in the light adaptive processes of the visual system. The sole source of retinal dopamine is dopaminergic amacrine cells (DACs). We and others have previously demonstrated that DACs are activated by rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) upon illumination. However, it is still not clear how each class of photosensitive cells generates light responses in DACs. We genetically isolated cone function in mice to specifically examine the cone-mediated responses of DACs and their neural pathways. In addition to the reported excitatory input to DACs from light-increment (ON) bipolar cells, we found that cones alternatively signal to DACs via a retrograde signalling pathway from ipRGCs. Cones also produce ON and light-decrement (OFF) inhibitory responses in DACs, which are mediated by other amacrine cells, likely driven by type 1 and type 2/3a OFF bipolar cells, respectively. Dye injections indicated that DACs had similar morphological profiles with or without ON/OFF inhibition. Our data demonstrate that cones utilize specific parallel excitatory and inhibitory circuits to modulate DAC activity and efficiently regulate dopamine release and the light-adaptive state of the retina.

A Rapid Cell Expansion Process for Production of Engineered Autologous CAR-T Cell Therapies.

Science.gov (United States)

Lu, Tangying Lily; Pugach, Omar; Somerville, Robert; Rosenberg, Steven A; Kochenderfer, James N; Better, Marc; Feldman, Steven A

2016-12-01

The treatment of B-cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials. 1-6 The anti-CD19 CAR-T cell production method used to support initial trials relied on numerous manual, open process steps, human serum, and 10 days of cell culture to achieve a clinical dose. 7 This approach limited the ability to support large multicenter clinical trials, as well as scale up for commercial cell production. Therefore, studies were completed to streamline and optimize the original National Cancer Institute production process by removing human serum from the process in order to minimize the risk of viral contamination, moving process steps from an open system to functionally closed system operations in order to minimize the risk of microbial contamination, and standardizing additional process steps in order to maximize process consistency. This study reports a procedure for generating CD19 CAR-T cells in 6 days, using a functionally closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process.

Tumorigenicity studies for human pluripotent stem cell-derived products.

Science.gov (United States)

Kuroda, Takuya; Yasuda, Satoshi; Sato, Yoji

2013-01-01

Human pluripotent stem cells (hPSCs), i.e. human embryonic stem cells and human induced pluripotent stem cells, are able to self-renew and differentiate into multiple cell types. Because of these abilities, numerous attempts have been made to utilize hPSCs in regenerative medicine/cell therapy. hPSCs are, however, also tumorigenic, that is, they can give rise to the progressive growth of tumor nodules in immunologically unresponsive animals. Therefore, assessing and managing the tumorigenicity of all final products is essential in order to prevent ectopic tissue formation, tumor development, and/or malignant transformation elicited by residual pluripotent stem cells after implantation. No detailed guideline for the tumorigenicity testing of hPSC-derived products has yet been issued for regenerative medicine/cell therapy, despite the urgent necessity. Here, we describe the current situations and issues related to the tumorigenicity testing of hPSC-derived products and we review the advantages and disadvantages of several types of tumorigenicity-associated tests. We also refer to important considerations in the execution and design of specific studies to monitor the tumorigenicity of hPSC-derived products.

Continued SOFC cell and stack technology and improved production methods

Energy Technology Data Exchange (ETDEWEB)

Wandel, M.; Brodersen, K.; Phair, J. (and others)

2009-05-15

Within this project significant results are obtained on a number of very diverse areas ranging from development of cell production, metallic creep in interconnect to assembling and test of stacks with foot print larger than 500 cm2. Out of 38 milestones 28 have been fulfilled and 10 have been partly fulfilled. This project has focused on three main areas: 1) The continued cell development and optimization of manufacturing processes aiming at production of large foot-print cells, improving cell performance and development environmentally more benign production methods. 2) Stack technology - especially stacks with large foot print and improving the stack design with respect to flow geometry and gas leakages. 3) Development of stack components with emphasis on sealing (for 2G as well as 3G), interconnect (coat, architecture and creep) and test development. Production of cells with a foot print larger than 500 cm2 is very difficult due to the brittleness of the cells and great effort has been put into this topic. Eight cells were successfully produced making it possible to assemble and test a real stack thereby giving valuable results on the prospects of stacks with large foot print. However, the yield rate is very low and a significant development to increase this yield lies ahead. Several lessons were learned on the stack level regarding 'large foot print' stacks. Modelling studies showed that the width of the cell primarily is limited by production and handling of the cell whereas the length (in the flow direction) is limited by e.g. pressure drop and necessary manifolding. The optimal cell size in the flow direction was calculated to be between approx20 cm and < 30 cm. From an economical point of view the production yield is crucial and stacks with large foot print cell area are only feasible if the cell production yield is significantly enhanced. Co-casting has been pursued as a production technique due to the possibilities in large scale production

Human umbilical cord mesenchymal stem cells increase interleukin-9 production of CD4+ T cells

Science.gov (United States)

Yang, Zhou Xin; Chi, Ying; Ji, Yue Ru; Wang, You Wei; Zhang, Jing; Luo, Wei Feng; Li, Li Na; Hu, Cai Dong; Zhuo, Guang Sheng; Wang, Li Fang; Han, Zhi-Bo; Han, Zhong Chao

2017-01-01

Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhanced IL-9 production by CD4+ T cells. Transwell experiments suggested that UC-MSC promotion of IL-9 production by CD4+ T cells was dependent on cell-cell contact. Upregulated expression of CD106 was observed in UC-MSC co-cultured with CD4+ T cells, and the addition of a blocking antibody of CD106 significantly impaired the ability of UC-MSC to promote IL-9 production by CD4+ T cells. Therefore, the results of the present study demonstrated that UC-MSC promoted the generation of IL-9 producing cells, which may be mediated, in part by CD106. The findings may act to expand understanding and knowledge of the immune modulatory role of UC-MSC. PMID:29042945

High-Volume Production of Lightweight Multijunction Solar Cells

Science.gov (United States)

Youtsey, Christopher

2015-01-01

MicroLink Devices, Inc., has transitioned its 6-inch epitaxial lift-off (ELO) solar cell fabrication process into a manufacturing platform capable of sustaining large-volume production. This Phase II project improves the ELO process by reducing cycle time and increasing the yield of large-area devices. In addition, all critical device fabrication processes have transitioned to 6-inch production tool sets designed for volume production. An emphasis on automated cassette-to-cassette and batch processes minimizes operator dependence and cell performance variability. MicroLink Devices established a pilot production line capable of at least 1,500 6-inch wafers per month at greater than 80 percent yield. The company also increased the yield and manufacturability of the 6-inch reclaim process, which is crucial to reducing the cost of the cells.

Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro.

OpenAIRE

O'REILLY, VINCENT

2013-01-01

PUBLISHED Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4(+), CD8α(+), and ...

Direct fuel cell product design improvement

Energy Technology Data Exchange (ETDEWEB)

Maru, H.C.; Farooque, M. [Energy Research Corp., Danbury, CT (United States)

1996-12-31

Significant milestones have been attained towards the technology development field testing and commercialization of direct fuel cell power plant since the 1994 Fuel Cell Seminar. Under a 5-year cooperative agreement with the Department of Energy signed in December 1994, Energy Research Corporation (ERC) has been developing the design for a MW-scale direct fuel cell power plant with input from previous technology efforts and the Santa Clara Demonstration Project. The effort encompasses product definition in consultation with the Fuel Cell Commercialization Group, potential customers, as well as extensive system design and packaging. Manufacturing process improvements, test facility construction, cell component scale up, performance and endurance improvements, stack engineering, and critical balance-of-plant development are also addressed. Major emphasis of this product design improvement project is on increased efficiency, compactness and cost reduction to establish a competitive place in the market. A 2.85 MW power plant with an efficiency of 58% and a footprint of 420 m{sup 2} has been designed. Component and subsystem testing is being conducted at various levels. Planning and preparation for verification of a full size prototype unit are in progress. This paper presents the results obtained since the last fuel cell seminar.

Tetherin restricts productive HIV-1 cell-to-cell transmission.

Directory of Open Access Journals (Sweden)

Nicoletta Casartelli

2010-06-01

Full Text Available The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24 impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

Fuel Production from Seawater and Fuel Cells Using Seawater.

Science.gov (United States)

Fukuzumi, Shunichi; Lee, Yong-Min; Nam, Wonwoo

2017-11-23

Seawater is the most abundant resource on our planet and fuel production from seawater has the notable advantage that it would not compete with growing demands for pure water. This Review focuses on the production of fuels from seawater and their direct use in fuel cells. Electrolysis of seawater under appropriate conditions affords hydrogen and dioxygen with 100 % faradaic efficiency without oxidation of chloride. Photoelectrocatalytic production of hydrogen from seawater provides a promising way to produce hydrogen with low cost and high efficiency. Microbial solar cells (MSCs) that use biofilms produced in seawater can generate electricity from sunlight without additional fuel because the products of photosynthesis can be utilized as electrode reactants, whereas the electrode products can be utilized as photosynthetic reactants. Another important source for hydrogen is hydrogen sulfide, which is abundantly found in Black Sea deep water. Hydrogen produced by electrolysis of Black Sea deep water can also be used in hydrogen fuel cells. Production of a fuel and its direct use in a fuel cell has been made possible for the first time by a combination of photocatalytic production of hydrogen peroxide from seawater and dioxygen in the air and its direct use in one-compartment hydrogen peroxide fuel cells to obtain electric power. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fission product detection by means of photovoltaic cells

International Nuclear Information System (INIS)

Liatard, E.; Akrouf, S.; Bruandet, J.F.; Fontenille, A.; Glasser, F.; Stassi, P.; Tsan Ung Chan

1988-01-01

The response of photovoltaic cells to heavy ions and fission products have been tested in-beam. Their main advantages are their extremely low price, their low sensitivity to energetic light ions with respect to fission products, and the possibility to cut and fit them together to any shape without dead zone. The time output signals of a charge sensitive preamplifier connected to these cells allows fast coincidences. A resolution of 12 ns (FWHM) has been measured between two cells. (orig.)

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells.

Science.gov (United States)

Da Silva, Daniel; Lachaud, Christophe; Cotelle, Valérie; Brière, Christian; Grat, Sabine; Mazars, Christian; Thuleau, Patrice

2011-05-01

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium dependent programmed cell death (PCD) in tobacco BY-2 cells. In addition, we have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction. 

Engineered CHO cells for production of diverse, homogeneous glycoproteins

DEFF Research Database (Denmark)

Yang, Zhang; Wang, Shengjun; Halim, Adnan

2015-01-01

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferas...

Cell culture media impact on drug product solution stability.

Science.gov (United States)

Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

2016-07-08

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016. © 2016 American Institute of Chemical Engineers.

miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

Science.gov (United States)

Fischer, Simon; Paul, Albert Jesuran; Wagner, Andreas; Mathias, Sven; Geiss, Melanie; Schandock, Franziska; Domnowski, Martin; Zimmermann, Jörg; Handrick, René; Hesse, Friedemann; Otte, Kerstin

2015-10-01

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells. © 2015 Wiley Periodicals, Inc.

A roadmap for cost-of-goods planning to guide economic production of cell therapy products.

Science.gov (United States)

Lipsitz, Yonatan Y; Milligan, William D; Fitzpatrick, Ian; Stalmeijer, Evelien; Farid, Suzanne S; Tan, Kah Yong; Smith, David; Perry, Robert; Carmen, Jessica; Chen, Allen; Mooney, Charles; Fink, John

2017-12-01

Cell therapy products are frequently developed and produced without incorporating cost considerations into process development, contributing to prohibitively costly products. Herein we contextualize individual process development decisions within a broad framework for cost-efficient therapeutic manufacturing. This roadmap guides the analysis of cost of goods (COG) arising from tissue procurement, material acquisition, facility operation, production, and storage. We present the specific COG considerations related to each of these elements as identified through a 2013 International Society for Cellular Therapy COG survey, highlighting the differences between autologous and allogeneic products. Planning and accounting for COG at each step in the production process could reduce costs, allowing for more affordable market pricing to improve the long-term viability of the cell therapy product and facilitate broader patient access to novel and transformative cell therapies. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Recombinant Protein Production and Insect Cell Culture and Process

Science.gov (United States)

Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

1997-01-01

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

78 FR 49528 - Consolidation of Wound Care Products Containing Live Cells

Science.gov (United States)

2013-08-14

...] Consolidation of Wound Care Products Containing Live Cells AGENCY: Food and Drug Administration, HHS. ACTION... certain wound care products containing live cells from the Center for Devices and Radiological Health... CDRH and CBER. FDA believes that as more wound care products containing live cells are developed such...

Merkel cells are long-lived cells whose production is stimulated by skin injury✰

Science.gov (United States)

Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

2017-01-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

Merkel cells are long-lived cells whose production is stimulated by skin injury.

Science.gov (United States)

Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

2017-02-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of blood products on inflammatory response in endothelial cells in vitro.

Directory of Open Access Journals (Sweden)

Martin Urner

Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

Production of solar photovoltaic cells on the Moon

Science.gov (United States)

Criswell, David R.; Ignatiev, Alex

1991-01-01

Solar energy is directly available on the sunward lunar surface. Most, if not all, the materials are available on the Moon to make silicon based solar photovoltaic cells. A few additional types are possible. There is a small but growing literature on production of lunar derived solar cells. This literature is reviewed. Topics explored include trade-offs of local production versus import of key materials, processing options, the scale and nature of production equipment, implications of storage requirements, and the end-uses of the energy. Directions for future research and demonstrations are indicated.

Glycoengineering of CHO Cells to Improve Product Quality.

Science.gov (United States)

Wang, Qiong; Yin, Bojiao; Chung, Cheng-Yu; Betenbaugh, Michael J

2017-01-01

Chinese hamster ovary (CHO) cells represent the predominant platform in biopharmaceutical industry for the production of recombinant biotherapeutic proteins, especially glycoproteins. These glycoproteins include oligosaccharide or glycan attachments that represent one of the principal components dictating product quality. Especially important are the N-glycan attachments present on many recombinant glycoproteins of commercial interest. Furthermore, altering the glycan composition can be used to modulate the production quality of a recombinant biotherapeutic from CHO and other mammalian hosts. This review first describes the glycosylation network in mammalian cells and compares the glycosylation patterns between CHO and human cells. Next genetic strategies used in CHO cells to modulate the sialylation patterns through overexpression of sialyltransfereases and other glycosyltransferases are summarized. In addition, other approaches to alter sialylation including manipulation of sialic acid biosynthetic pathways and inhibition of sialidases are described. Finally, this review also covers other strategies such as the glycosylation site insertion and manipulation of glycan heterogeneity to produce desired glycoforms for diverse biotechnology applications.

CHO On A Detox: Removing By-Product Formation Through Cell Engineering

DEFF Research Database (Denmark)

Pereira, Sara; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

Chinese Hamster Ovary (CHO) cells are the preferred hosts for the production of therapeutic glycoproteins. However, there is a need for improvement of the bioprocesses towards increased cell growth and higher productivities without compromising the product quality. Efforts to obtain tailor-made p......-made products with the desired properties that meet the requirements of regulatory authorities are continuously being made. Of equal relevance is to develop methods to engineer cell lines with improved by-product metabolism....

[Product safety analysis of somatic cell cloned bovine].

Science.gov (United States)

Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

2010-05-01

Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

Science.gov (United States)

Lin, Yi-Chu; Wang, Po-Yu; Tsai, Shih-Chih; Lin, Chien-Liang; Tai, Hsuen-Yung; Lo, Chi-Fang; Wu, Shiow-Ing; Chiang, Yu-Mei; Liu, Li-Ling

2015-01-01

Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients.

Topography of ganglion cell production in the cat's retina

International Nuclear Information System (INIS)

Walsh, C.; Polley, E.H.

1985-01-01

The ganglion cells of the cat's retina form several classes distinguishable in terms of soma size, axon diameter, dendritic morphology, physiological properties, and central connections. Labeling with [ 3 H]thymidine shows that the ganglion cells which survive in the adult are produced as several temporally shifted, overlapping waves: medium-sized cells are produced before large cells, whereas the smallest ganglion cells are produced throughout the period of ganglion cell generation. Large cells and medium-sized cells show the same distinctive pattern of production, forming rough spirals around the area centralis. The oldest cells tend to lie superior and nasal to the area centralis, whereas cells in the inferior nasal retina and inferior temporal retina are, in general, progressively younger. Within each retinal quadrant, cells nearer the area centralis tend to be older than cells in the periphery, but there is substantial overlap. The retinal raphe divides the superior temporal quadrant into two zones with different patterns of cell addition. Superior temporal retina near the vertical meridian adds cells only slightly later than superior nasal retina, whereas superior temporal retina near the horizontal meridian adds cells very late, contemporaneously with inferior temporal retina. The broader wave of production of smaller ganglion cells seems to follow this same spiral pattern at its beginning and end. The presence of the area centralis as a nodal point about which ganglion cell production in the retinal quadrants pivots suggests that the area centralis is already an important retinal landmark even at the earliest stages of retinal development

Rod photoreceptors express GPR55 in the adult vervet monkey retina

DEFF Research Database (Denmark)

Bouskila, Joseph; Javadi, Pasha; Casanova, Christian

2013-01-01

. Yet, its formal classification is still a matter of debate. CB1R and CB2R expression patterns are well described for rodent and monkey retinas. In the monkey retina, CB1R has been localized in its neural (cone photoreceptor, horizontal, bipolar, amacrine and ganglion cells) and CB2R in glial...

Simulation-based comparisons of four apparel cell production modes of one clothing production line

Directory of Open Access Journals (Sweden)

Guoqiang Pan

2014-10-01

Full Text Available Purpose: This research, by using the SIMIO simulation platform, provides a quantitative and comparative analysis of how the efficiency of four different cell production modes is affected. It is hoped that the outcomes will be of some help for garment factories to optimize their production lines. Design/methodology/approach: The SIMIO simulation platform was employed in the research and comparisons were made of the simulation test results about the four different production modes. Findings: The operation mode, number of operators, and number of buffer areas are key factors affecting the production line efficiency, and need to be reasonably set to achieve the highest efficiency. Originality/value: As most research literature so far is qualitative, this research provided a simulation-based quantitative analysis of the production efficiency under different cell production modes.

Stem-cell-derived products: an FDA update.

Science.gov (United States)

Moos, Malcolm

2008-12-01

The therapeutic potential of products derived from stem cells of various types has prompted increasing research and development and public attention. Initiation of human clinical trials in the not-too-distant future is now a realistic possibility. It is, therefore, important to weigh the potential benefits against known, theoretical and totally unsuspected risks in light of current knowledge to ensure that subjects participating in these trials are afforded the most reasonable balance possible between potential risks and potential benefits. There are no apparent differences in fundamental, qualitative biological characteristics between stem-cell-derived products and other cellular therapies regulated by the United States Food and Drug Administration (FDA). Existing authorities can, therefore, be applied. Nevertheless, these products do have properties that require careful evaluation.

the production of mouse embryonic stem cells

Indian Academy of Sciences (India)

MADU

What history tells us VII. Twenty-five years ago: the production of mouse embryonic stem cells ... cells into the cavity of the blastocyst, it will be possible to test the effect of .... to the use of efficient immunosuppressive drugs like cyclosporin – was ...

Immunoglobulin production induced in vitro by glucocorticoid hormones: T cell-dependent stimulation of immunoglobulin production without B cell proliferation in cultures of human peripheral blood lymphocytes

International Nuclear Information System (INIS)

Grayson, J.; Dooley, N.J.; Koski, I.R.; Blaese, R.M.

1981-01-01

The direct effects of steroid hormones on the production of immunoglobulins and DNA synthesis by human T and B lymphocytes was evaluated in cultures of peripheral blood mononuclear cells. As detected by a reverse hemolytic plaque assay, the addition of 0.1 mM to 10 nM hydrocortisone to lymphocytes in culture in the absence of other stimulants or mitogens, resulted in the dramatic induction of immunoglobulin production with responses comparable to those seen in similar cultures stimulated with pokeweed mitogen. Steroid-stimulated immunoglobulin production was first seen after 48 h and peaked at 8-10 d of culture. The production of IgG, IgA, and IgM was induced following incubation with steroid. Glucocorticoids, but not estrogens or androgens, were capable of mediating this effect, and only compounds with affinity for the glucocorticoid receptor were active. The induction of immunoglobulin production was dependent on both T cells and monocytes; cultures depleted of either cell type did not produce immunoglobulin when stimulated with glucocorticoid hormones. Proliferation of B cells or T cells could not be detected by [/sup 3/H]thymidine incorporation or total cell recovery from steroid-stimulated cultures, even though such cultures demonstrated marked increases in immunoglobulin production. The mechanism responsible for this functional maturation of B cells to become high rate immunoglobulin producing cells is as yet undefined, although it appears to involve more than merely steroid mediated inactivation of suppressor T cells

Multi-stage high cell continuous fermentation for high productivity and titer.

Science.gov (United States)

Chang, Ho Nam; Kim, Nag-Jong; Kang, Jongwon; Jeong, Chang Moon; Choi, Jin-dal-rae; Fei, Qiang; Kim, Byoung Jin; Kwon, Sunhoon; Lee, Sang Yup; Kim, Jungbae

2011-05-01

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.

T cell Ig domain and mucin domain 1 engagement on invariant NKT cells in the presence of TCR stimulation enhances IL-4 production but inhibits IFN-gamma production.

Science.gov (United States)

Kim, Hye Sung; Kim, Hyun Soo; Lee, Chang Woo; Chung, Doo Hyun

2010-04-15

The T cell Ig domain and mucin domain (TIM)1 protein expressed on the surface of Th2 cells regulates the immune response by modulating cytokine production. However, the functional roles of TIM1 have not been examined in NKT cells. Therefore, we investigated the immunologic effects of TIM1 on NKT cells. We found that mouse NK1.1(+)TCR-beta(+), alpha-galactosyl ceramide/CD1d dimer(+) NKT, and NKT hybridoma (DN32.D3) cells constitutively express TIM1 and TIM4 on their surface. Engagement of TIM1 on NKT cells by any of several anti-TIM1 mAbs suppressed the production of IFN-gamma in the presence of TCR stimulation in vitro and in vivo, whereas the effects of such engagement on Th2 cytokine production by the NKT cells varied with the particular anti-TIM1 Ab clone. Moreover, in DN32.D3 TIM4-knockdown NKT hybridoma cells, TIM1 engagement by rTIM1 or TIM4 enhanced IL-4 production while inhibiting IFN-gamma production in the presence of alpha-galactosyl ceramide stimulation. TIM1 engagement increased GATA-3 expression but reduced T-bet expression in NKT cells in the presence of TCR engagement. The adoptive transfer of NKT cells preincubated with anti-TIM1 mAbs into Jalpha18(-/-) mice aggravated bleomycin-induced pulmonary fibrosis by suppressing IFN-gamma production. Taken together, these results suggest that TIM1 costimulation on NKT cells enhances the cellular production of IL-4 while inhibiting the production of IFN-gamma. Thus, as a differential regulator of the immune response, TIM1 on NKT cells may be a useful therapeutic target for immune diseases.

Microbial reverse-electrodialysis chemical-production cell for acid and alkali production

KAUST Repository

Zhu, Xiuping

2013-06-01

A new type of bioelectrochemical system, called a microbial reverse-electrodialysis chemical-production cell (MRCC), was developed to produce acid and alkali using energy derived from organic matter (acetate) and salinity gradients (NaCl solutions representative of seawater and river water). A bipolar membrane (BPM) was placed next to the anode to prevent Cl- contamination and acidification of the anolyte, and to produce protons for HCl recovery. A 5-cell paired reverse-electrodialysis (RED) stack provided the electrical energy required to overcome the BPM over-potential (0.3-0.6 V), making the overall process spontaneous. The MRCC reactor produced electricity (908 mW/m2) as well as concentrated acidic and alkaline solutions, and therefore did not require an external power supply. After a fed-batch cycle, the pHs of the chemical product solutions were 1.65 ± 0.04 and 11.98 ± 0.10, due to the production of 1.35 ± 0.13 mmol of acid, and 0.59 ± 0.14 mmol of alkali. The acid- and alkali-production efficiencies based on generated current were 58 ± 3% and 25 ± 3%. These results demonstrated proof-of-concept acid and alkali production using only renewable energy sources. © 2013 Elsevier B.V.

Organic and Inorganic Nitrogen Impact Chlorella variabilis Productivity and Host Quality for Viral Production and Cell Lysis.

Science.gov (United States)

Cheng, Yu-Shen; Labavitch, John; VanderGheynst, Jean S

2015-05-01

Microalgae have been proposed as a potential feedstock for biofuel production; however, cell disruption is usually required for collection and utilization of cytoplasmic polysaccharides and lipids. Virus infection might be one approach to disrupt the cell wall. The concentration of yeast extract and presence of KNO3 in algae cultivation media were investigated to observe their effects on Chlorella variabilis NC64A physiology and composition and the subsequent effect on production of Chlorella virus and disruption of infected cells. Cytoplasmic starch accumulation increased from 5% to approximately 35% of the total dry weight when yeast extract decreased from 1 to 0.25 g L(-1). When cells were cultured with the lowest nitrogen levels, the total polysaccharide accounted for more than 50% of the cell wall, which was 1.7 times higher than the content in cells cultured with the highest nitrogen levels. The C/N ratio of the algal biomass decreased by a factor of approximately 2 when yeast extract increased from 0.25 to 1 g L(-1). After virus infection, cells with a low C/N ratio produced a 7.6 times higher burst size than cells with a high C/N ratio, suggesting that the nitrogen content in C. variabilis has a large influence on viral production and cell lysis. The results have implications on management of nitrogen for both the synthesis of products from algae and product recovery via viral lysis.

A Pixel-Encoder Retinal Ganglion Cell with Spatially Offset Excitatory and Inhibitory Receptive Fields

Directory of Open Access Journals (Sweden)

Keith P. Johnson

2018-02-01

Full Text Available The spike trains of retinal ganglion cells (RGCs are the only source of visual information to the brain. Here, we genetically identify an RGC type in mice that functions as a pixel encoder and increases firing to light increments (PixON-RGC. PixON-RGCs have medium-sized dendritic arbors and non-canonical center-surround receptive fields. From their receptive field center, PixON-RGCs receive only excitatory input, which encodes contrast and spatial information linearly. From their receptive field surround, PixON-RGCs receive only inhibitory input, which is temporally matched to the excitatory center input. As a result, the firing rate of PixON-RGCs linearly encodes local image contrast. Spatially offset (i.e., truly lateral inhibition of PixON-RGCs arises from spiking GABAergic amacrine cells. The receptive field organization of PixON-RGCs is independent of stimulus wavelength (i.e., achromatic. PixON-RGCs project predominantly to the dorsal lateral geniculate nucleus (dLGN of the thalamus and likely contribute to visual perception.

TIM-1 signaling in B cells regulates antibody production

International Nuclear Information System (INIS)

Ma, Juan; Usui, Yoshihiko; Takeda, Kazuyoshi; Harada, Norihiro; Yagita, Hideo; Okumura, Ko; Akiba, Hisaya

2011-01-01

Highlights: → TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. → Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. → TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3 + anti-CD28-stimulated CD4 + T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

TIM-1 signaling in B cells regulates antibody production

Energy Technology Data Exchange (ETDEWEB)

Ma, Juan [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Usui, Yoshihiko [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Ophthalmology, Tokyo Medical University, 6-7-1 Nishi-shinjuku-ku, Tokyo 160-0023 (Japan); Takeda, Kazuyoshi [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Harada, Norihiro [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Respiratory Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Research Institute for Diseases of Old Ages, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Yagita, Hideo; Okumura, Ko [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Akiba, Hisaya, E-mail: hisaya@juntendo.ac.jp [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

2011-03-11

Highlights: {yields} TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. {yields} Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. {yields} TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3{sup +} anti-CD28-stimulated CD4{sup +} T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

Engineered mammalian cells for production of recombinant proteins

DEFF Research Database (Denmark)

2017-01-01

The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

Tobacco and e-cigarette products initiate Kupffer cell inflammatory responses.

Science.gov (United States)

Rubenstein, David A; Hom, Sarah; Ghebrehiwet, Berhane; Yin, Wei

2015-10-01

Kupffer cells are liver resident macrophages that are responsible for screening and clearing blood of pathogens and foreign particles. It has recently been shown that Kupffer cells interact with platelets, through an adhesion based mechanism, to aid in pathogen clearance and then these platelets re-enter the general systemic circulation. Thus, a mechanism has been identified that relates liver inflammation to possible changes in the systemic circulation. However, the role that Kupffer cells play in cardiovascular disease initiation/progression has not been elucidated. Thus, our objective was to determine whether or not Kupffer cells are responsive to a classical cardiovascular risk factor and if these changes can be transmitted into the general systemic circulation. If Kupffer cells initiate inflammatory responses after exposure to classical cardiovascular risk factors, then this provides a potential alternative/synergistic pathway for cardiovascular disease initiation. We aimed to elucidate the prevalence of this potential pathway. We hypothesized that Kupffer cells would initiate a robust inflammatory response after exposure to tobacco cigarette or e-cigarette products and that the inflammatory response would have the potential to antagonize other salient cells for cardiovascular disease progression. To test this, Kupffer cells were incubated with tobacco smoke extracts, e-cigarette vapor extracts or pure nicotine. Complement deposition onto Kupffer cells, Kupffer cell complement receptor expression, oxidative stress production, cytokine release and viability and density were assessed after the exposure. We observed a robust inflammatory response, oxidative stress production and cytokine release after Kupffer cells were exposed to tobacco or e-cigarette extracts. We also observed a marginal decrease in cell viability coupled with a significant decrease in cell density. In general, this was not a function of the extract formulation (e.g. tobacco vs. e

Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture.

Science.gov (United States)

Shimizu, Takashi; Kaji, Ayami; Murayama, Chiaki; Magata, Fumie; Shirasuna, Koumei; Wakamiya, Kaori; Okuda, Kiyoshi; Miyamoto, Akio

2012-01-01

Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48 h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48 h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

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Say Kong Ng

2013-04-01

Full Text Available From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX amplification technology or the glutamine synthetase (GS system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

Host cell proteins in biotechnology-derived products: A risk assessment framework.

Science.gov (United States)

de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

2015-11-01

To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

Regulatory Oversight of Cell and Gene Therapy Products in Canada.

Science.gov (United States)

Ridgway, Anthony; Agbanyo, Francisca; Wang, Jian; Rosu-Myles, Michael

2015-01-01

Health Canada regulates gene therapy products and many cell therapy products as biological drugs under the Canadian Food and Drugs Act and its attendant regulations. Cellular products that meet certain criteria, including minimal manipulation and homologous use, may be subjected to a standards-based approach under the Safety of Human Cells, Tissues and Organs for Transplantation Regulations. The manufacture and clinical testing of cell and gene therapy products (CGTPs) presents many challenges beyond those for protein biologics. Cells cannot be subjected to pathogen removal or inactivation procedures and must frequently be administered shortly after final formulation. Viral vector design and manufacturing control are critically important to overall product quality and linked to safety and efficacy in patients through concerns such as replication competence, vector integration, and vector shedding. In addition, for many CGTPs, the value of nonclinical studies is largely limited to providing proof of concept, and the first meaningful data relating to appropriate dosing, safety parameters, and validity of surrogate or true determinants of efficacy must come from carefully designed clinical trials in patients. Addressing these numerous challenges requires application of various risk mitigation strategies and meeting regulatory expectations specifically adapted to the product types. Regulatory cooperation and harmonisation at an international level are essential for progress in the development and commercialisation of these products. However, particularly in the area of cell therapy, new regulatory paradigms may be needed to harness the benefits of clinical progress in situations where the resources and motivation to pursue a typical drug product approval pathway may be lacking.

Bags versus flasks: a comparison of cell culture systems for the production of dendritic cell-based immunotherapies.

Science.gov (United States)

Fekete, Natalie; Béland, Ariane V; Campbell, Katie; Clark, Sarah L; Hoesli, Corinne A

2018-04-19

In recent years, cell-based therapies targeting the immune system have emerged as promising strategies for cancer treatment. This review summarizes manufacturing challenges related to production of antigen presenting cells as a patient-tailored cancer therapy. Understanding cell-material interactions is essential because in vitro cell culture manipulations to obtain mature antigen-producing cells can significantly alter their in vivo performance. Traditional antigen-producing cell culture protocols often rely on cell adhesion to surface-treated hydrophilic polystyrene flasks. More recent commercial and investigational cancer immunotherapy products were manufactured using suspension cell culture in closed hydrophobic fluoropolymer bags. The shift to closed cell culture systems can decrease risks of contamination by individual operators, as well as facilitate scale-up and automation. Selecting closed cell culture bags over traditional open culture systems entails different handling procedures and processing controls, which can affect product quality. Changes in culture vessels also entail changes in vessel materials and geometry, which may alter the cell microenvironment and resulting cell fate decisions. Strategically designed culture systems will pave the way for the generation of more sophisticated and highly potent cell-based cancer vaccines. As an increasing number of cell-based therapies enter the clinic, the selection of appropriate cell culture vessels and materials becomes a critical consideration that can impact the therapeutic efficacy of the product, and hence clinical outcomes and patient quality of life. © 2018 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Do cell based tissue engineering products for meniscus regeneration influence vascularization?

Science.gov (United States)

Koch, Matthias; Ehrenreich, Tobias; Koehl, Gudrun; Pattappa, Girish; Pfeifer, Christian; Loibl, Markus; Müller, Michael; Nerlich, Michael; Angele, Peter; Zellner, Johannes

2017-01-01

Meniscus regeneration is observed within the peripheral, vascularized zone but decreases in the inner two thirds alongside the vascularization. Within this avascular area, cell-based tissue-engineering-approaches appear to be a promising strategy for the treatment of meniscal defects. Evaluation of the angiogenic potential of cell-based tissue-engineering-products for meniscus healing. Evaluation of angiogenesis induced by rabbit meniscus-pellets, meniscus-cells (MC) or mesenchymal stem-cells (MSC) in cell-based tissue-engineering-products within a rabbit meniscus-ring was performed using a transparent dorsal skin fold chamber in nude mice. Observations were undertaken during a 14 days period. Cell preconditioning differed between experimental groups. Immunohistochemical analysis of the regenerated tissue in the meniscus-ring induced by cell loaded composite scaffolds for differentiation and anti-angiogenic factors were performed. Meniscus-pellets and MSC-/MC-based tissue-engineering-products induced angiogenesis. An accelerated vascularization was detected in the group of meniscus-pellets derived from the vascularized zone compared to avascular meniscus-pellets. In terms of cell-based tissue-engineering-products, chondrogenic preconditioning resulted in significantly increased vessel growth. MSC-constructs showed an accelerated angiogenesis. Immunohistochemical evaluation showed a progressive differentiation and lower content for anti-angiogenic endostatin in the precultured group. Preconditioning of MC-/MSC-based tissue-engineering-products is a promising tool to influence the angiogenic potential of tissue-engineering-products and to adapt these properties according to the aimed tissue qualities.

Production status of GaAs/Ge solar cells and panels

Science.gov (United States)

Smith, B.; Gillanders, M.; Vijayakumar, P.; Lillington, D.; Yang, H.; Rolph, R.

1991-01-01

GaAs/Ge solar cells with lot average efficiencies in excess of 18 percent were produced by MOCVD growth techniques. A description of the cell, its performance and the production facility are discussed. Production GaAs/Ge cells of this type were recently assembled into circuits and bonded to aluminum honeycomb panels to be used as the solar array for the British UOSAT-F program.

JSI-124 inhibits IgE production in an IgE B cell line

International Nuclear Information System (INIS)

Cui, Lulu; Bi, Jiacheng; Yan, Dehong; Ye, Xiufeng; Zheng, Mingxing; Yu, Guang; Wan, Xiaochun

2017-01-01

IgE is a key effector molecule in atopic diseases; however, the regulation mechanisms of IgE production in IgE B cells remain poorly understood. In the present study, we demonstrate that JSI-124 (cucurbitacin I), a selective STAT3 inhibitor, selectively inhibits production of IgE by a human IgE B cell line, CRL-8033 cells, while does not affect the IgG production by IgG B cell lines. In the aspect of molecular mechanism, we found that Igλ, but not Ighe, gene expression was suppressed by JSI-124. The above effects of JSI-124 were not mediated by affecting cellular proliferation or apoptosis. Furthermore, multiple B cell differentiation-related genes expression was not significantly affected by JSI-124. Taken together, we demonstrate a potential strategy of therapeutically suppressing IgE production without affecting IgG production in atopic patients. - Highlights: • JSI-124 inhibits IgE production in an IgE B cell line, CRL-8033 cells. • JSI-124 does not affect IgG production by IgG B cell lines. • JSI-124 inhibits IgE production mainly by suppressing transcription of Igλ.

Simple electrolytic cell for production of elemental fluorine

International Nuclear Information System (INIS)

Dides F, M.; Padilla S, U.

1990-01-01

It was constructed and tested a simple electrolytic cell for the production of elemental fluorine. The fluorine production is essential in the obtainment of uranium hexafluoride, a compound for the nuclear fuel cycle. (A.C.A.S.)

Proinflammatory interleukins' production by adipose tissue-derived mesenchymal stromal cells: the impact of cell culture conditions and cell-to-cell interaction.

Science.gov (United States)

Andreeva, Elena; Andrianova, Irina; Rylova, Julia; Gornostaeva, Aleksandra; Bobyleva, Polina; Buravkova, Ludmila

2015-08-01

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL-6 and IL-8 production by adipose-derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O2 concentrations, some highly up-regulated at hypoxia. IL-6 and IL-8 production was inversely dependent on cell culture density. In early (first-third) passages, IL-6 and IL-8 concentration was higher at 20% O2 and in late (8th-12th) passages under 5% O2. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL-6 and did not result in the elevation of IL-8 concentration. Thereby, the production of proinflammatory interleukins (IL-6 and IL-8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood-borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. SIGNIFICANCE PARAGRAPH: Ex vivo expansion is widely used for increasing the number of adipose-derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science. Copyright © 2015 John Wiley & Sons, Ltd.

Pilot production of 4 sq cm ITO/InP photovoltaic solar cells

Science.gov (United States)

Gessert, T. A.; Li, X.; Coutts, T. J.; Tzafaras, N.

1991-01-01

Experimental results of a pilot production of 32 4-sq cm indium tin oxide (ITO)InP space solar cells are presented. The discussion includes analysis of the device performance of the best cells produced as well as the performance range of all production cells. The experience gained from the production is discussed, indicating other issues that may be encountered when large-scale productions are initiated. Available data on a 4-sq cm ITO/InP cell that was flown on the UoSAT-5 satellite is reported.

Automated computation of arbor densities: a step toward identifying neuronal cell types

Directory of Open Access Journals (Sweden)

Uygar eSümbül

2014-11-01

Full Text Available The shape and position of a neuron convey information regarding its molecular and functional identity. The identification of cell types from structure, a classic method, relies on the time-consuming step of arbor tracing. However, as genetic tools and imaging methods make data-driven approaches to neuronal circuit analysis feasible, the need for automated processing increases. Here, we first establish that mouse retinal ganglion cell types can be as precise about distributing their arbor volumes across the inner plexiform layer as they are about distributing the skeletons of the arbors. Then, we describe an automated approach to computing the spatial distribution of the dendritic arbors, or arbor density, with respect to a global depth coordinate based on this observation. Our method involves three-dimensional reconstruction of neuronal arbors by a supervised machine learning algorithm, post-processing of the enhanced stacks to remove somata and isolate the neuron of interest, and registration of neurons to each other using automatically detected arbors of the starburst amacrine interneurons as fiducial markers. In principle, this method could be generalizable to other structures of the CNS, provided that they allow sparse labeling of the cells and contain a reliable axis of spatial reference.

Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development

Directory of Open Access Journals (Sweden)

N. Tolosa de Talamoni

2010-05-01

Full Text Available The immunolocalization of the plasma membrane calcium pump (PMCA was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody 5F10 from human erythrocyte plasma membrane crossreacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP 333 immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.

Generation of a KOR-Cre knockin mouse strain to study cells involved in kappa opioid signaling.

Science.gov (United States)

Cai, Xiaoyun; Huang, Huizhen; Kuzirian, Marissa S; Snyder, Lindsey M; Matsushita, Megumi; Lee, Michael C; Ferguson, Carolyn; Homanics, Gregg E; Barth, Alison L; Ross, Sarah E

2016-01-01

The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non-neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR-Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR-Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR-Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR-Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR-Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR. © 2015 Wiley Periodicals, Inc.

Erythroid cells in vitro: from developmental biology to blood transfusion products.

Science.gov (United States)

Migliaccio, Anna Rita; Whitsett, Carolyn; Migliaccio, Giovanni

2009-07-01

Red blood cells (RBCs) transfusion plays a critical role in numerous therapies. Disruption of blood collection by political unrest, natural disasters and emerging infections and implementation of restrictions on the use of erythropoiesis-stimulating agents in cancer may impact blood availability in the near future. These considerations highlight the importance of developing alternative blood products. Knowledge about the processes that control RBC production has been applied to the establishment of culture conditions allowing ex-vivo generation of RBCs in numbers close to those (2.5 x 10 cells/ml) present in a transfusion, from cord blood, donated blood units or embryonic stem cells. In addition, experimental studies demonstrate that such cells protect mice from lethal bleeding. Therefore, erythroid cells generated ex vivo may be suitable for transfusion provided they can be produced safely in adequate numbers. However, much remains to be done to translate a theoretical production of approximately 2.5 x 10 RBCs in the laboratory into a 'clinical grade production process'. This review summarizes the state-of-the-art in establishing ex-vivo culture conditions for erythroid cells and discusses the most compelling issues to be addressed to translate this progress into a clinical grade transfusion product.

Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Science.gov (United States)

Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S.

2017-01-01

ABSTRACT Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28112405

Microbial reverse-electrodialysis chemical-production cell for acid and alkali production

KAUST Repository

Zhu, Xiuping; Hatzell, Marta C.; Cusick, Roland D.; Logan, Bruce E.

2013-01-01

A new type of bioelectrochemical system, called a microbial reverse-electrodialysis chemical-production cell (MRCC), was developed to produce acid and alkali using energy derived from organic matter (acetate) and salinity gradients (NaCl solutions

Synthetic yeast based cell factories for vanillin-glucoside production

DEFF Research Database (Denmark)

Strucko, Tomas

and controlled expression/overexpression of genes of interest. De novo biosynthetic pathway for vanillin-β-glucoside production was employed as a model system for several case studies in this project. In order to construct yeast cell factories fulfilling current demands of industrial biotechnology, methods......The yeast Saccharomyces cerevisiae is well a characterized microorganism and widely used as eukaryotic model organism as well as a key cell factory for bioproduction of various products. The latter comprise a large variety of scientifically and industrially relevant products such as low-value bulk...... chemicals and biofuels, food additives, high-value chemicals and recombinant proteins. Despite the recent achievements in the fields of systems biology and metabolic engineering together with availability of broad genetic engineering toolbox, the full potential of S. cerevisiae as a cell factory is not yet...

Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

Science.gov (United States)

Kamachi, Yasuharu; Omasa, Takeshi

2018-04-01

Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells.

Science.gov (United States)

Dreesen, Imke A J; Fussenegger, Martin

2011-04-01

Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over-expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess-relevant characteristics of Chinese hamster ovary (CHO) cell-derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell-cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub-optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR-transgenic CHO-derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four-fold increase compared to the parental production cell line. mTOR-based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Copyright © 2010 Wiley Periodicals, Inc.

Cell-based product classification procedure: What can be done differently to improve decisions on borderline products?

Science.gov (United States)

Izeta, Ander; Herrera, Concha; Mata, Rosario; Astori, Giuseppe; Giordano, Rosaria; Hernández, Carmen; Leyva, Laura; Arias, Salvador; Oyonarte, Salvador; Carmona, Gloria; Cuende, Natividad

2016-07-01

In June 2015, European Medicines Agency/Committee for Advanced Therapies (CAT) released the new version of the reflection paper on classification of advanced therapy medicinal products (ATMPs) established to address questions of borderline cases in which classification of a product based on genes, cells or tissues is unclear. The paper shows CAT's understanding of substantial manipulation and essential function(s) criteria that define the legal scope of cell-based medicinal products. This article aims to define the authors' viewpoint on the reflection paper. ATMP classification has intrinsic weaknesses derived from the lack of clarity of the evolving concepts of substantial manipulation and essential function(s) as stated in the EU Regulation, leading to the risk of differing interpretations and misclassification. This might result in the broadening of ATMP scope at the expense of other products such as cell/tissue transplants and blood products, or even putting some present and future clinical practice at risk of being classified as ATMP. Because of the major organizational, economic and regulatory implications of product classification, we advocate for increased interaction between CAT and competent authorities (CAs) for medicines, blood and blood components and tissues and cells or for the creation of working groups including representatives of all parties as recently suggested by several CAs. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

Directory of Open Access Journals (Sweden)

Takashi Kawaguchi

1999-01-01

Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

Grain dust induces IL-8 production from bronchial epithelial cells: the effect of dexamethasone on IL-8 production.

Science.gov (United States)

Park, H S; Suh, J H; Kim, H Y; Kwon, O J; Choi, D C

1999-04-01

Recent publications have suggested an active participation of neutrophils to induce bronchoconstriction after inhalation of grain dust (GD). To further understand the role of neutrophils in the pathogenesis of GD-induced asthma, this investigation was designed to determine whether human bronchial epithelial cells could produce IL-8 production and to observe the effect of dexamethasone on IL-8 production. We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four concentrations (1 to 200 microg/mL) of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant, which was derived from the culture of PBMC from a GD-induced asthmatic subject under the exposure to 10 microg/mL of GD, and compared with those cultured without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. To evaluate the effect of dexamethasone on IL-8 production, four concentrations (5 to 5000 ng/mL) of dexamethasone were pre-incubated for 24 hours and the same experiments were repeated. There was significant production of IL-8 from bronchial epithelial cells with additions of GD in a dose-dependent manner (P < .05), which was significantly augmented with additions of PBMC supernatant (P < .05) at each concentration. Compared with the untreated sample, pretreatment of dexamethasone could induced a remarkable inhibitions (15% to 55%) of IL-8 production from bronchial epithelial cells in a dose-dependent manner. These results suggest that IL-8 production from bronchial epithelial cells may contribute to neutrophil recruitment occurring in GD-induced airway inflammation. The downregulation of IL-8 production by dexamethasone from bronchial epithelial cells may contribute to the efficacy of this compound in

Pigment Production Analysis in Human Melanoma Cells.

Science.gov (United States)

Hopkin, Amelia Soto; Paterson, Elyse K; Ruiz, Rolando; Ganesan, Anand K

2016-05-25

The human epidermal melanocyte is a highly specialized pigmented cell that serves to protect the epidermis from ultraviolet (UV) damage through the production of melanin, or melanogenesis. Misregulation in melanogenesis leading to either hyper- or hypo-pigmentation is found in human diseases such as malasma and vitiligo. Current therapies for these diseases are largely unsuccessful and the need for new therapies is necessary. In order to identify genes and or compounds that can alter melanogenesis, methods are required that can detect changes in pigment production as well as expression of key melanogenesis transcription factors and enzymes. Here we describe methods to detect changes in melanogenesis in a human melanoma cell line, MNT-1, by (1) analyzing pigment production by measuring the absorbance of melanin present by spectrophotometry, (2) analyzing transcript expression of potent regulators of melanogenesis by qunatitative reverse-transcription (RT)PCR and (3) analyzing protein expression of potent regulators of melanogenesis by Western blot (WB).

Human umbilical cord-derived mesenchymal stem cells utilise Activin-A to suppress Interferon-gamma production by natural killer cells.

Directory of Open Access Journals (Sweden)

Debanjana eChaterjee

2014-12-01

Full Text Available Following allogeneic hematopoietic stem cell transplantation (HSCT, interferon (IFN-gamma levels in the recipient’s body can strongly influence the clinical outcome. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs are lucrative as biological tolerance-inducers in HSCT settings. Hence, we studied the molecular mechanism of how UC-MSCs influence natural killer (NK cell-mediated IFN-gamma production. Allogeneic NK cells were cultured in direct contact with UC-MSCs or cell free supernatants from MSC cultures (MSC conditioned media. We found that soluble factors secreted by UC-MSCs strongly suppressed IL-12/IL-18-induced IFN-gamma production by NK cells by reducing phosphorylation of STAT4, NF-kB as well as T-bet activity. UC-MSCs secreted considerable amounts of Activin-A, which could suppress IFN-gamma production by NK cells. Neutralisation of Activin-A in MSC-conditioned media significantly abrogated their suppressive abilities. Till date, multiple groups have reported that prostaglandin (PG-E2 produced by MSCs can suppress NK cell functions. Indeed, we found that inhibition of PGE2 production by MSCs could also significantly restore IFN-gamma production. However, the effects of Activin-A and PGE2 were not cumulative. To the best of our knowledge, we are first to report the role of Activin-A in MSC-mediated suppression of IFN-gamma production by NK cells.

Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

Directory of Open Access Journals (Sweden)

Lohr Verena

2011-09-01

Full Text Available Abstract Background Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA and infectious viral particle (IVP titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 106 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products. Results To further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 106 cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 1011 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 1015 IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles. Conclusions These results

Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production.

Science.gov (United States)

Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S; Polizzi, Karen M

2017-06-01

Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole-cell biosensors. Biotechnol. Bioeng. 2017;114: 1290-1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Production of alkaline protease by Teredinobacter turnirae cells ...

African Journals Online (AJOL)

The conditions for immobilizing the new alkaline protease-producing bacteria strain Teredinobacter turnirae by entrapment in calcium alginate gel were investigated. The influence of alginate concentration (20, 25 and 30 g/l) and initial cell loading (ICL) on enzyme production were studied. The production of alkaline ...

Analysis of cell line variation in biochemical production of protoporphyrin IX

Science.gov (United States)

Gibbs, Summer L.; Chen, Bin; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

2006-02-01

Protoporphyrin IX (PpIX) is produced via the heme synthesis pathway by the cell following administration of aminolevulinic acid (ALA). ALA synthase, the enzyme that produces ALA in the cell from glycine and succinyl-coenzyme A, is inhibited in a feedback mechanism by heme and thus is the rate limiting enzyme in the heme synthesis pathway. Since ALA is administered systemically, the rate limiting step that naturally exists in the cells is bypassed, however it is currently unclear why cells have different rate limiting steps in the ALA-PpIX synthesis pathway, and more specifically which types of cancer cells are most productive. It has been determined that when the same amount of ALA is administered to a wide panel of cancer cells in vitro that vastly differing amounts of PpIX are produced. The steps for the ALA-PpIX pathway occur in and around the mitochondria of the cell, but interestingly no correlation is seen between PpIX production and mitochondrial content of the cell, following ALA administration. However, total cell area shows positive correlation with PpIX production. Administration of the iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed and thus increases the detectable amount of PpIX in each cell line. The cell lines that have the lowest PpIX production following administration of ALA alone show the largest increase in production following the combined administration of ALA and L1. PpIX fluorescence is thought to be a measure of cellular activity and the goal of the current study was to determine which cell lines would be the most promising targets for fluorescence detection or monitoring response to therapy. The results indicate that the cells with larger size and larger numbers of mitochondria may be good potential targets for this therapy. While this conclusion may appear obvious, it is not universally true, and cellular specific

Methods for production of proteins in host cells

Science.gov (United States)

Donnelly, Mark; Joachimiak, Andrzej

2004-01-13

The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

Benchmarking of commercially available CHO cell culture media for antibody production.

Science.gov (United States)

Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

2015-06-01

In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability.

Science.gov (United States)

Cai, Zhenzhen; Kastell, Anja; Speiser, Claire; Smetanska, Iryna

2013-09-01

The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

Development of High-Productivity Continuous Ethanol Production using PVA-Immobilized Zymomonas mobilis in an Immobilized-Cells Fermenter

Directory of Open Access Journals (Sweden)

Nurhayati Nurhayati

2015-07-01

Full Text Available Ethanol as one of renewable energy was being considered an excellent alternative clean-burning fuel to replace gasoline. Continuous ethanol fermentation systems had offered important economic advantages compared to traditional systems. Fermentation rates were significantly improved, especially when continuous fermentation was integrated with cell immobilization techniques to enrich the cells concentration in fermentor. Growing cells of Zymomonas mobilis immobilized in polyvinyl alcohol (PVA gel beads were employed in an immobilized-cells fermentor for continuous ethanol fermentation from glucose. The glucose loading, dilution rate, and cells loading were varied in order to determine which best condition employed in obtaining both high ethanol production and low residual glucose with high dilution rate. In this study, 20 g/L, 100 g/L, 125 g/L and 150 g/L of glucose concentration and 20% (w/v, 40% (w/v and 50% (w/v of cells loading were employed with range of dilution rate at 0.25 to 1 h-1. The most stable production was obtained for 25 days by employing 100 g/L of glucose loading. Meanwhile, the results also exhibited that 125 g/L of glucose loading as well as 40% (w/v of cells loading yielded high ethanol concentration, high ethanol productivity, and acceptable residual glucose at 62.97 g/L, 15.74 g/L/h and 0.16 g/L, respectively. Furthermore, the dilution rate of 4 hour with 100 g/L and 40% (w/v of glucose and cells loading was considered as the optimum condition with ethanol production, ethanol productivity and residual glucose obtained were 49.89 g/L, 12.47 g/L/h, and 2.04 g/L, respectively. This recent study investigated ethanol inhibition as well. The present research had proved that high sugar concentration was successfully converted to ethanol. These achieved results were promising for further study.

Effect of failures and repairs on multiple cell production lines

Energy Technology Data Exchange (ETDEWEB)

Legato, P.; Bobbio, A.; Roberti, L.

1989-01-01

This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis

DEFF Research Database (Denmark)

Hall, Vanessa Jane; Jacobsen, J.; Gunnarsson, A.

2011-01-01

Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis......Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis...

ApoER2 Function in the Establishment and Maintenance of Retinal Synaptic Connectivity

Science.gov (United States)

Trotter, Justin H.; Klein, Martin; Jinwal, Umesh K.; Abisambra, Jose F.; Dickey, Chad A.; Tharkur, Jeremy; Masiulis, Irene; Ding, Jindong; Locke, Kirstin G.; Rickman, Catherine Bowes; Birch, David G.; Weeber, Edwin J.; Herz, Joachim

2011-01-01

The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult ApoER2 was expressed by A-II amacrine cells. ApoER2 knockout (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding disabled-1 (Dab1) and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway while the alternatively-spliced exon19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity. PMID:21976526

Tumor necrosis factor alpha production in irradiated cells in vitro

International Nuclear Information System (INIS)

Koeteles, G.J.; Bognar, G.; Kubasova, T.

1994-01-01

Normal and tumor cell lines were used to investigate tumor necrosis factor (TNFα) production and its radiation sensitivity. The cells were irradiated with gamma rays using different doses from 0.25 Gy up to 5 Gy. The number of plated cells, changes of proliferation and TNFα production were determined during the following four post-irradiation days. For TNFα quantity measurement immuno-radiometric assay (IRMA) and enzyme amplified sensitivity assay (EASIA) was used. The results suggest that though gamma irradiation decreased cell proliferation in a dose dependent manner, the quantity produced in the post-irradiation period increased considerably in each irradiated sample. (N.T.) 3 refs.; 2 figs.; 1 tab

Development of a yeast cell factory for production of aromatic products

DEFF Research Database (Denmark)

Rodriguez Prado, Edith Angelica; Kildegaard, Kanchana Rueksomtawin; Li, Mingji

2014-01-01

There is much interest in aromatic chemicals in the chemical industry as these can be used for production of dyes, anti-oxidants, nutraceuticals and food ingredients. Yeast is a widely used cell factory and it is particularly well suited for production of aromatic chemicals via complex biosynthetic...... routes involving P450 enzymes. In Saccharomyces cerevisiae the fluxes towards aromatic acids (L-tryptophan, L-tyrosine and L-phenylalanine) are strictly controlled on transcriptional and kinetic levels and therefore are difficult to manipulate. We engineered S. cerevisiae for increased production...... of aromatic compounds by eliminating degradation, up-regulating the key enzyme encoding genes, and removing feed-back inhibition in the pathway. In order to test the strain performance we overexpressed heterologous pathway for coumaric acid production. We obtained 4-fold higher concentrations of coumaric acid...

Interleukin-8 stimulates progesterone production via the MEK pathway in ovarian theca cells.

Science.gov (United States)

Shimizu, Takashi; Imamura, Eri; Magata, Fumie; Murayama, Chiaki; Miyamoto, Akio

2013-02-01

Interleukin 8 (IL-8) is a chemoattractant associated with ovulation in the mammalian ovary. This chemokine is also involved in the recruitment and activation of neutrophils. Using bovine tissue, we examined the possible role of IL-8 in steroid production by theca cells of the large ovarian follicles. IL-8 promoted progesterone production and stimulated StAR expression in cultured theca cells. The inhibitor of p38 did not disturb the P4 production and StAR expression in IL-8-treated theca cells. On the other hand, the inhibitor of MEK disturbed the P4 production and expression of StAR in theca cells treated with IL-8. These results suggest that IL-8 is associated with progesterone production in bovine theca cells via the MEK pathway.

Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Liu, Zihe; Hou, Jin; Martinez Ruiz, José Luis

2013-01-01

.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner...... turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h−1 that influences protein production. Thus, for lower specific growth rates, the α-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas......With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e...

Recombinant protein production from stable mammalian cell lines and pools.

Science.gov (United States)

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell

KAUST Repository

Wang, Aijie; Sun, Dan; Cao, Guangli; Wang, Haoyu; Ren, Nanqi; Wu, Wei-Min; Logan, Bruce E.

2011-01-01

Hydrogen gas production from cellulose was investigated using an integrated hydrogen production process consisting of a dark fermentation reactor and microbial fuel cells (MFCs) as power sources for a microbial electrolysis cell (MEC). Two MFCs

Host cells and methods for production of isobutanol

Science.gov (United States)

Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

2017-10-17

Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

Energy Technology Data Exchange (ETDEWEB)

H.C. Maru; M. Farooque

2003-03-01

The program efforts are focused on technology and system optimization for cost reduction, commercial design development, and prototype system field trials. The program is designed to advance the carbonate fuel cell technology from full-size field test to the commercial design. FuelCell Energy, Inc. (FCE) is in the later stage of the multiyear program for development and verification of carbonate fuel cell based power plants supported by DOE/NETL with additional funding from DOD/DARPA and the FuelCell Energy team. FCE has scaled up the technology to full-size and developed DFC{reg_sign} stack and balance-of-plant (BOP) equipment technology to meet product requirements, and acquired high rate manufacturing capabilities to reduce cost. FCE has designed submegawatt (DFC300A) and megawatt (DFC1500 and DFC3000) class fuel cell products for commercialization of its DFC{reg_sign} technology. A significant progress was made during the reporting period. The reforming unit design was optimized using a three-dimensional stack simulation model. Thermal and flow uniformities of the oxidant-In flow in the stack module were improved using computational fluid dynamics based flow simulation model. The manufacturing capacity was increased. The submegawatt stack module overall cost was reduced by {approx}30% on a per kW basis. An integrated deoxidizer-prereformer design was tested successfully at submegawatt scale using fuels simulating digester gas, coal bed methane gas and peak shave (natural) gas.

Sorting cells of the microalga Chlorococcum littorale with increased triacylglycerol productivity.

Science.gov (United States)

Cabanelas, Iago Teles Dominguez; van der Zwart, Mathijs; Kleinegris, Dorinde M M; Wijffels, René H; Barbosa, Maria J

2016-01-01

Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity. 1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1-S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l(-1) day(-1) (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l(-1) day(-1)). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs. All combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale, without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production.

Minimal RED Cell Pairs Markedly Improve Electrode Kinetics and Power Production in Microbial Reverse Electrodialysis Cells

KAUST Repository

Cusick, Roland D.

2013-12-17

Power production from microbial reverse electrodialysis cell (MRC) electrodes is substantially improved compared to microbial fuel cells (MFCs) by using ammonium bicarbonate (AmB) solutions in multiple RED cell pair stacks and the cathode chamber. Reducing the number of RED membranes pairs while maintaining enhanced electrode performance could help to reduce capital costs. We show here that using only a single RED cell pair (CP), created by operating the cathode in concentrated AmB, dramatically increased power production normalized to cathode area from both acetate (Acetate: from 0.9 to 3.1 W/m 2-cat) and wastewater (WW: 0.3 to 1.7 W/m2), by reducing solution and charge transfer resistances at the cathode. A second RED cell pair increased RED stack potential and reduced anode charge transfer resistance, further increasing power production (Acetate: 4.2 W/m2; WW: 1.9 W/m2). By maintaining near optimal electrode power production with fewer membranes, power densities normalized to total membrane area for the 1-CP (Acetate: 3.1 W/m2-mem; WW: 1.7 W/m2) and 2-CP (Acetate: 1.3 W/m2-mem; WW: 0.6 W/m2) reactors were much higher than previous MRCs (0.3-0.5 W/m2-mem with acetate). While operating at peak power, the rate of wastewater COD removal, normalized to reactor volume, was 30-50 times higher in 1-CP and 2-CP MRCs than that in a single chamber MFC. These findings show that even a single cell pair AmB RED stack can significantly enhance electrical power production and wastewater treatment. © 2013 American Chemical Society.

Minimal RED Cell Pairs Markedly Improve Electrode Kinetics and Power Production in Microbial Reverse Electrodialysis Cells

KAUST Repository

Cusick, Roland D.; Hatzell, Marta; Zhang, Fang; Logan, Bruce E.

2013-01-01

Power production from microbial reverse electrodialysis cell (MRC) electrodes is substantially improved compared to microbial fuel cells (MFCs) by using ammonium bicarbonate (AmB) solutions in multiple RED cell pair stacks and the cathode chamber. Reducing the number of RED membranes pairs while maintaining enhanced electrode performance could help to reduce capital costs. We show here that using only a single RED cell pair (CP), created by operating the cathode in concentrated AmB, dramatically increased power production normalized to cathode area from both acetate (Acetate: from 0.9 to 3.1 W/m 2-cat) and wastewater (WW: 0.3 to 1.7 W/m2), by reducing solution and charge transfer resistances at the cathode. A second RED cell pair increased RED stack potential and reduced anode charge transfer resistance, further increasing power production (Acetate: 4.2 W/m2; WW: 1.9 W/m2). By maintaining near optimal electrode power production with fewer membranes, power densities normalized to total membrane area for the 1-CP (Acetate: 3.1 W/m2-mem; WW: 1.7 W/m2) and 2-CP (Acetate: 1.3 W/m2-mem; WW: 0.6 W/m2) reactors were much higher than previous MRCs (0.3-0.5 W/m2-mem with acetate). While operating at peak power, the rate of wastewater COD removal, normalized to reactor volume, was 30-50 times higher in 1-CP and 2-CP MRCs than that in a single chamber MFC. These findings show that even a single cell pair AmB RED stack can significantly enhance electrical power production and wastewater treatment. © 2013 American Chemical Society.

The inhibition of superoxide production in EL4 lymphoma cells overexpressing growth hormone.

Science.gov (United States)

Arnold, Robyn E; Weigent, Douglas A

2003-05-01

A substantial body of research exists to support the production of growth hormone by cells of the immune system. However, the function and mechanism of action of lymphocyte-derived growth hormone remain largely unelucidated. Since, it has been found that exogenous growth hormone (GH) primes neutrophils for the production of reactive oxygen intermediates (ROI) and in particular superoxide (O2-), we investigated the role of GH on the production of O2- in T cells. Furthermore, we examined whether endogenous and exogenous GH act similarly. Our studies show that overexpression of GH in EL4, a T-cell lymphoma cell line, results in a decrease in the production of O2- compared to control cells, as detected using the fluorescent dye, dihydroethidium. O2- production in control cells was not affected by treatment with inhibitors of xanthine oxidase or a non-specific NADPH-oxidase inhibitor. However, treatment with diallyl sulfide, an inhibitor of cytochrome P450 2E1 mimicked the reduction in O2- production seen in cells overexpressing GH. Although no significant change could be detected in CYP2E1 protein levels, CYP2E1 activity was found to be greater in control EL4 than in cells overexpressing GH. Both the decrease in O2- production and the lower CYP2E1 activity in GH overexpressing cells could be abrogated by treatment with N(G)-monomethyl-L-arginine, an inhibitor of nitric oxide synthase. The overexpression of GH protects cells from apoptosis induced by isoniazid, a CYP2E1 inducer, suggesting a role for nitric oxide as a mediator in the regulation of xenobiotic metabolism and apoptosis-protection by lymphocyte GH.

B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers.

Science.gov (United States)

Fukazawa, Yoshinori; Lum, Richard; Okoye, Afam A; Park, Haesun; Matsuda, Kenta; Bae, Jin Young; Hagen, Shoko I; Shoemaker, Rebecca; Deleage, Claire; Lucero, Carissa; Morcock, David; Swanson, Tonya; Legasse, Alfred W; Axthelm, Michael K; Hesselgesser, Joseph; Geleziunas, Romas; Hirsch, Vanessa M; Edlefsen, Paul T; Piatak, Michael; Estes, Jacob D; Lifson, Jeffrey D; Picker, Louis J

2015-02-01

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

Directory of Open Access Journals (Sweden)

Mitchell Brianna M

2003-04-01

Full Text Available Abstract Background Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP, an antagonist of KCNA but not KCNQ channels. Methods Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3 and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA and steroidogenic acute regulatory protein (StAR was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. Results 4-AP (2 mM decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production

Method for reducing ammonium and lactate production in cho cells

DEFF Research Database (Denmark)

2018-01-01

The present invention relates to modified producer cells for improved production of therapeutic proteins. Specifically, the inventors have found that removing genes involved in amino acid catabolism in Chinese Hamster Ovary (CHO) cells improves the cell growth and viability and likely also...

Robo signaling regulates the production of cranial neural crest cells.

Science.gov (United States)

Li, Yan; Zhang, Xiao-Tan; Wang, Xiao-Yu; Wang, Guang; Chuai, Manli; Münsterberg, Andrea; Yang, Xuesong

2017-12-01

Slit/Robo signaling plays an important role in the guidance of developing neurons in developing embryos. However, it remains obscure whether and how Slit/Robo signaling is involved in the production of cranial neural crest cells. In this study, we examined Robo1 deficient mice to reveal developmental defects of mouse cranial frontal and parietal bones, which are derivatives of cranial neural crest cells. Therefore, we determined the production of HNK1 + cranial neural crest cells in early chick embryo development after knock-down (KD) of Robo1 expression. Detection of markers for pre-migratory and migratory neural crest cells, PAX7 and AP-2α, showed that production of both was affected by Robo1 KD. In addition, we found that the transcription factor slug is responsible for the aberrant delamination/EMT of cranial neural crest cells induced by Robo1 KD, which also led to elevated expression of E- and N-Cadherin. N-Cadherin expression was enhanced when blocking FGF signaling with dominant-negative FGFR1 in half of the neural tube. Taken together, we show that Slit/Robo signaling influences the delamination/EMT of cranial neural crest cells, which is required for cranial bone development. Copyright © 2017. Published by Elsevier Inc.

Human Lung Mast Cell Products Regulate Airway Smooth Muscle CXCL10 Levels.

Science.gov (United States)

Alkhouri, H; Cha, V; Tong, K; Moir, L M; Armour, C L; Hughes, J M

2014-01-01

In asthma, the airway smooth muscle (ASM) produces CXCL10 which may attract CXCR3(+) mast/T cells to it. Our aim was to investigate the effects of mast cell products on ASM cell CXCL10 production. ASM cells from people with and without asthma were stimulated with IL-1 β , TNF- α , and/or IFN γ and treated with histamine (1-100  μ M) ± chlorpheniramine (H1R antagonist; 1  μ M) or ranitidine (H2R antagonist; 50  μ M) or tryptase (1 nM) ± leupeptin (serine protease inhibitor; 50  μ M), heat-inactivated tryptase, or vehicle for 4 h or 24 h. Human lung mast cells (MC) were isolated and activated with IgE/anti-IgE and supernatants were collected after 2 h or 24 h. The supernatants were added to ASM cells for 48 h and ASM cell CXCL10 production detected using ELISA (protein) and real-time PCR (mRNA). Histamine reduced IL-1 β /TNF- α -induced CXCL10 protein, but not mRNA, levels independent of H1 and H2 receptor activation, whereas tryptase and MC 2 h supernatants reduced all cytokine-induced CXCL10. Tryptase also reduced CXCL10 levels in a cell-free system. Leupeptin inhibited the effects of tryptase and MC 2 h supernatants. MC 24 h supernatants contained TNF- α and amplified IFN γ -induced ASM cell CXCL10 production. This is the first evidence that MC can regulate ASM cell CXCL10 production and its degradation. Thus MC may regulate airway myositis in asthma.

Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

Science.gov (United States)

Ströh, Sebastian; Puller, Christian; Swirski, Sebastian; Hölzel, Maj-Britt; van der Linde, Lea I S; Segelken, Jasmin; Schultz, Konrad; Block, Christoph; Monyer, Hannah; Willecke, Klaus; Weiler, Reto; Greschner, Martin; Janssen-Bienhold, Ulrike; Dedek, Karin

2018-02-21

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light

Electrochemical characterisation of solid oxide cell electrodes for hydrogen production

DEFF Research Database (Denmark)

Bernuy-Lopez, Carlos; Knibbe, Ruth; He, Zeming

2011-01-01

Oxygen electrodes and steam electrodes are designed and tested to develop improved solid oxide electrolysis cells for H2 production with the cell support on the oxygen electrode. The electrode performance is evaluated by impedance spectroscopy testing of symmetric cells at open circuit voltage (OCV...

Studies on the mechanism of endogenous pyrogen production. II. Role of cell products in the regulation of pyrogen release from blood leukocytes.

Science.gov (United States)

Bodel, P

1974-09-01

Some characteristics of the process by which endogenous pyrogen (EP), the mediator of fever, is released from cells were examined by using human blood leukocytes incubated in vitro. Studies were designed to examine a possible role for leukocyte products, including EP, in the induction, augmentation, or suppression of pyrogen release by blood leukocytes. Products of stimulated leukocytes, including a partially purified preparation of EP, did not induce significant activation of nonstimulated cells. Also, no evidence was obtained that stimulated cell products either augment or inhibit pyrogen production by other stimulated cells. A feedback control of EP production was thus not observed. A crude preparation of EP, containing other products of activated cells, maintained its pyrogenicity when incubated at pH 7.4 but not at pH 5.0. These studies thus provide no support for hypothesized control mechanisms regulating production of EP by blood leukocytes. By contrast, local inactivation of EP at inflammatory sites may modify the amount of EP entering the blood, and hence fever.

The relationship of metabolic burden to productivity levels in CHO cell lines.

Science.gov (United States)

Zou, Wu; Edros, Raihana; Al-Rubeai, Mohamed

2018-03-01

The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Fed-batch CHO cell culture for lab-scale antibody production

DEFF Research Database (Denmark)

Fan, Yuzhou; Ley, Daniel; Andersen, Mikael Rørdam

2017-01-01

Fed-batch culture is the most commonly used upstream process in industry today for recombinant monoclonal antibody production using Chinese hamster ovary cells. Developing and optimizing this process in the lab is crucial for establishing process knowledge, which enable rapid and predictable tech......-transfer to manufacturing scale. In this chapter, we will describe stepwise how to carry out fed-batch CHO cell culture for lab-scale antibody production....

Bipolar cell gap junctions serve major signaling pathways in the human retina.

Science.gov (United States)

Kántor, Orsolya; Varga, Alexandra; Nitschke, Roland; Naumann, Angela; Énzsöly, Anna; Lukáts, Ákos; Szabó, Arnold; Németh, János; Völgyi, Béla

2017-08-01

Connexin36 (Cx36) constituent gap junctions (GJ) throughout the brain connect neurons into functional syncytia. In the retina they underlie the transmission, averaging and correlation of signals prior conveying visual information to the brain. This is the first study that describes retinal bipolar cell (BC) GJs in the human inner retina, whose function is enigmatic even in the examined animal models. Furthermore, a number of unique features (e.g. fovea, trichromacy, midget system) necessitate a reexamination of the animal model results in the human retina. Well-preserved postmortem human samples of this study are allowed to identify Cx36 expressing BCs neurochemically. Results reveal that both rod and cone pathway interneurons display strong Cx36 expression. Rod BC inputs to AII amacrine cells (AC) appear in juxtaposition to AII GJs, thus suggesting a strategic AII cell targeting by rod BCs. Cone BCs serving midget, parasol or koniocellular signaling pathways display a wealth of Cx36 expression to form homologously coupled arrays. In addition, they also establish heterologous GJ contacts to serve an exchange of information between parallel signaling streams. Interestingly, a prominent Cx36 expression was exhibited by midget system BCs that appear to maintain intimate contacts with bistratified BCs serving other pathways. These findings suggest that BC GJs in parallel signaling streams serve both an intra- and inter-pathway exchange of signals in the human retina.

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

Directory of Open Access Journals (Sweden)

Benson Heather L

2012-03-01

Full Text Available Abstract Background Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs and plasmacytoid (pDCs are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown. Methods Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d prior to transplanting into C57BL/6 mice (H-2b, followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+. Results Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production. Conclusion Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells.

Science.gov (United States)

Magata, F; Horiuchi, M; Miyamoto, A; Shimizu, T

2014-08-01

Uterine bacterial infection perturbs uterine and ovarian functions in postpartum dairy cows. Peptidoglycan (PGN) produced by gram-positive bacteria has been shown to disrupt the ovarian function in ewes. The aim of this study was to determine the effect of PGN on steroid production in bovine theca cells at different stages of follicular development. Bovine theca cells isolated from pre- and post-selection ovarian follicles (8.5mm in diameter, respectively) were cultured in vitro and challenged with PGN. Steroid production was evaluated by measuring progesterone (P4) and androstenedione (A4) concentration in culture media after 48 h or 96 h of culture. Bovine theca cells expressed PGN receptors including Toll-like receptor 2 and nucleotide-binding oligomerization domain 1 and 2. Treatment with PGN (1, 10, or 50 μg/ml) led to a decrease in P4 and A4 production by theca cells in both pre- and post-selection follicles. The mRNA expression of steroidogenic enzymes were decreased by PGN treatment. Moreover, A4 production was further suppressed when theca cells of post-selection follicles were simultaneously treated by PGN and lipopolysaccharide (0.1, 1, or 10 μg/ml). These findings indicate that bacterial toxins may act locally on ovarian steroidogenic cells and compromise follicular development in postpartum dairy cows. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hydrogen peroxide production is affected by oxygen levels in mammalian cell culture.

Science.gov (United States)

Maddalena, Lucas A; Selim, Shehab M; Fonseca, Joao; Messner, Holt; McGowan, Shannon; Stuart, Jeffrey A

2017-11-04

Although oxygen levels in the extracellular space of most mammalian tissues are just a few percent, under standard cell culture conditions they are not regulated and are often substantially higher. Some cellular sources of reactive oxygen species, like NADPH oxidase 4, are sensitive to oxygen levels in the range between 'normal' physiological (typically 1-5%) and standard cell culture (up to 18%). Hydrogen peroxide in particular participates in signal transduction pathways via protein redox modifications, so the potential increase in its production under standard cell culture conditions is important to understand. We measured the rates of cellular hydrogen peroxide production in some common cell lines, including C2C12, PC-3, HeLa, SH-SY5Y, MCF-7, and mouse embryonic fibroblasts (MEFs) maintained at 18% or 5% oxygen. In all instances the rate of hydrogen peroxide production by these cells was significantly greater at 18% oxygen than at 5%. The increase in hydrogen peroxide production at higher oxygen levels was either abolished or substantially reduced by treatment with GKT 137831, a selective inhibitor of NADPH oxidase subunits 1 and 4. These data indicate that oxygen levels experienced by cells in culture influence hydrogen peroxide production via NADPH oxidase 1/4, highlighting the importance of regulating oxygen levels in culture near physiological values. However, we measured pericellular oxygen levels adjacent to cell monolayers under a variety of conditions and with different cell lines and found that, particularly when growing at 5% incubator oxygen levels, pericellular oxygen was often lower and variable. Together, these observations indicate the importance, and difficulty, of regulating oxygen levels experienced by cells in culture. Copyright © 2017 Elsevier Inc. All rights reserved.

HAMCIND, Cell Burnup with Fission Products Poisoning

International Nuclear Information System (INIS)

Abe, Alfredo Y.; Dos Santos, Adimir

2002-01-01

1 - Description of program or function: HAMCIND is a cell burnup code based in a coupling between HAMMER-TECHNION and CINDER. The fission product poisoning is taken into account in an explicit fashion. 2 - Method of solution: The nonlinear coupled set of equations for the neutron transport and nuclide transmutation equations and nuclide transmutation equations in a unit cell is solved by HAMCIND in a quasi-static approach. The spectral transport equation is solved by HAMMER-TECHNION at the beginning of each time-step while the nuclide transmutation equations are solved by CINDER for every time-step. The HAMMER-TECHNION spectral calculations are performed taking into account the fission product contribution to the macroscopic cross sections (fast and thermal), in the inelastic scattering matrix and even in the thermal scattering matrices. 3 - Restrictions on the complexity of the problem: Restrictions and/or limitations for HAMCIND depend upon the local operating system

Muller glia, vision-guided ocular growth, retinal stem cells, and a little serendipity: the Cogan lecture.

Science.gov (United States)

Fischer, Andy J

2011-09-29

Hypothesis-driven science is expected to result in a continuum of studies and findings along a discrete path. By comparison, serendipity can lead to new directions that branch into different paths. Herein, I describe a diverse series of findings that were motivated by hypotheses, but driven by serendipity. I summarize how investigations into vision-guided ocular growth in the chick eye led to the identification of glucagonergic amacrine cells as key regulators of ocular elongation. Studies designed to assess the impact of the ablation of different types of neurons on vision-guided ocular growth led to the finding of numerous proliferating cells within damaged retinas. These proliferating cells were Müller glia-derived retinal progenitors with a capacity to produce new neurons. Studies designed to investigate Müller glia-derived progenitors led to the identification of a domain of neural stem cells that form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Accelerated ocular growth, caused by visual deprivation, stimulated the proliferation of CMZ progenitors. We formulated a hypothesis that growth-regulating glucagonergic cells may regulate both overall eye size (scleral growth) and the growth of the retina (proliferation of CMZ cells). Subsequent studies identified unusual types of glucagonergic neurons with terminals that ramify within the CMZ; these cells use visual cues to control equatorial ocular growth and the proliferation of CMZ cells. Finally, while studying the signaling pathways that stimulate CMZ and Müller glia-derived progenitors, serendipity led to the discovery of a novel type of glial cell that is scattered across the inner retinal layers.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

Science.gov (United States)

Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

2017-07-01

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Plutonium production in a remote cell

International Nuclear Information System (INIS)

Maddux, E.P.; Purcell, J.A.

1987-01-01

Production of 239 Pu metal has traditionally been carried out in glovebox enclosures for protection of operating personnel and the environment. With the advent of more stringent federal regulations for protecting personnel, the environment, and security of special nuclear materials, it is proposed that plutonium be processed in a totally remote environment using automated work stations and robotic materials transfer and equipment maintenance. At the Savannah River Plant, the existing plutonium production facility is a batch-type glovebox operation built in the 1950s. A viable alternative to restoration of the existing glovebox operation is to provide a facility that uses remote cell operation

Two different serum-free media and osmolality effect upon human 293 cell growth and adenovirus production.

Science.gov (United States)

Ferreira, Tiago B; Ferreira, Ana L; Carrondo, Manuel J T; Alves, Paula M

2005-11-01

Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells

International Nuclear Information System (INIS)

Reinsberg, Jochen; Wegener-Toper, Petra; Ven, Katrin van der; Ven, Hans van der; Klingmueller, Dietrich

2009-01-01

The phthalate ester mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of di-(2-ethylhexyl) phthalate, a high-production-volume chemical used as a plasticizer and solvent in numerous consumer products. MEHP has been demonstrated to be a reproductive toxicant in rodents decreasing estradiol and progesterone production in preovulatory granulosa cells. In the present study, we examined the effect of MEHP on steroid production of human granulosa-lutein (GL) cells. Human GL cells collected from women undergoing in vitro fertilization were cultured in medium containing FSH, hCG and 8-Br-cAMP, respectively, together with various concentrations of MEHP (0-500 μmol L -1 ). After incubation for 48 h estradiol and progesterone were assayed in the spent culture medium. Furthermore, aromatase activity and mRNA levels of GL cells were determined. Basal as well as FSH-, hCG- and 8-Br-cAMP-stimulated estradiol production of GL cells was suppressed by MEHP in a dose-dependent manner (IC 50 = 105 μmol L -1 , 138 μmol L -1 , 49 μmol L -1 and 78 μmol L -1 ). Furthermore aromatase activity and mRNA levels were reduced in GL cells cultured with MEHP. In contrast, MEHP did not alter the production of progesterone up to a concentration of 167 μmol L -1 . The present data indicate that MEHP is a specific inhibitor of estradiol production in human GL cells with a post-cAMP site of action. The inhibition of estradiol production obviously results from a reduction of aromatase activity on the transcript level. As the in vitro effective doses of MEHP are within the range of real environmental exposure levels an inhibitory effect on estrogen production in vivo seems to be possible.

Aloin Inhibits Interleukin (IL)-1β-Stimulated IL-8 Production in KB Cells.

Science.gov (United States)

Na, Hee Sam; Song, Yu Ri; Kim, Seyeon; Heo, Jun-Young; Chung, Hae-Young; Chung, Jin

2016-06-01

Interleukin (IL)-1β, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL-8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL-1β in IL-8 production and determines if aloin inhibits IL-1β-stimulated IL-8 production in epithelial cells. Saliva was collected from volunteers to determine IL-1β and IL-8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL-1β levels. KB cells were stimulated with IL-1β or saliva with or without IL-1 receptor agonist or specific mitogen-activated protein kinase (MAPK) inhibitors. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA). MAPK protein expression involved in IL-1β-induced IL-8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL-1β-induced IL-8 production was examined by ELISA and Western blot analysis. Saliva with high IL-1β strongly stimulated IL-8 production in KB cells, and IL-1 receptor agonist significantly inhibited IL-8 production. Low IL-1β-containing saliva did not increase IL-8 production. IL-1β treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor-kappa B. IL-1β-induced IL-8 production was decreased by p38 and extracellular signal-regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL-1β-induced IL-8 production in a dose-dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva-induced IL-8 production. Results indicated that IL-1β in saliva stimulates epithelial cells to produce IL-8 and that aloin effectively inhibits salivary IL-1β-induced IL-8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.

Heparin modulates human intestinal smooth muscle (HISM) cell proliferation and matrix production

International Nuclear Information System (INIS)

Graham, M.; Perr, H.; Drucker, D.E.; Diegelmann, R.F.

1986-01-01

(HISM) cell proliferation and collagen production may play a role in the pathogenesis of intestinal stricture in Crohn's disease. The present studies were performed to evaluate the effects of heparin, a known modulator of vascular smooth muscle cells, on HISM cell proliferation and collagen production. Heparin (100 μg/ml) was added daily to HISM cell cultures for cell proliferation studies and for 24 hours at various time points during culture for collagen synthesis studies. Collagen synthesis was determined by the uptake of 3 H proline into collagenase-sensitive protein. Heparin completely inhibited cell proliferation for 7 days, after which cell numbers increased but at a slower rate than controls. Cells released from heparin inhibition demonstrated catch-up growth to control levels. Collagen production was significantly inhibited by 24 hours exposure to heparin but only at those times during culture when collagen synthesis was maximal (8 to 12 days). Non-collagen protein synthesis was inhibited by heparin at all time points during culture. Heparin through its modulation of HISM cells may play an important role in the control of the extracellular matrix of the intestinal wall

Product quality considerations for mammalian cell culture process development and manufacturing.

Science.gov (United States)

Gramer, Michael J

2014-01-01

The manufacturing of a biologic drug from mammalian cells results in not a single substance, but an array of product isoforms, also known as variants. These isoforms arise due to intracellular or extracellular events as a result of biological or chemical modification. The most common examples related to biomanufacturing include amino acid modifications (glycosylation, isomerization, oxidation, adduct formation, pyroglutamate formation, phosphorylation, sulfation, amidation), amino acid sequence variants (genetic mutations, amino acid misincorporation, N- and C-terminal heterogeneity, clipping), and higher-order structure modifications (misfolding, aggregation, disulfide pairing). Process-related impurities (HCP, DNA, media components, viral particles) are also important quality attributes related to product safety. The observed ranges associated with each quality attribute define the product quality profile. A biologic drug must have a correct and consistent quality profile throughout clinical development and scale-up to commercial production to ensure product safety and efficacy. In general, the upstream process (cell culture) defines the quality of product-related substances, whereas the downstream process (purification) defines the residual level of process- and product-related impurities. The purpose of this chapter is to review the impact of the cell culture process on product quality. Emphasis is placed on studies with industrial significance and where the direct mechanism of product quality impact was determined. Where possible, recommendations for maintaining consistent or improved quality are provided.

Life cycle assessment of hydrogen production and fuel cell systems

International Nuclear Information System (INIS)

Dincer, I.

2007-01-01

This paper details life cycle assessment (LCA) of hydrogen production and fuel cell system. LCA is a key tool in hydrogen and fuel cell technologies for design, analysis, development; manufacture, applications etc. Energy efficiencies and greenhouse gases and air pollution emissions have been evaluated in all process steps including crude oil and natural gas pipeline transportation, crude oil distillation, natural gas reprocessing, wind and solar electricity generation , hydrogen production through water electrolysis and gasoline and hydrogen distribution and utilization

Air system in the hot cell for injectable radiopharmaceutical production: requirements for personnel and environment safety and protection of the product

Energy Technology Data Exchange (ETDEWEB)

Campos, Fabio E.; Araujo, Elaine B., E-mail: fecampos@ipen.b, E-mail: ebaraujo@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2009-07-01

Radiopharmaceuticals are applied in Nuclear Medicine in diagnostic and therapeutic procedures and must be manufactured in accordance with the basic principles of Good Manufacturing Practices (GMP) for sterile pharmaceutical products. In order to prevent the uncontrolled spread of radioactive contamination, the processing of radioactive materials requires an exhausted and shielded special enclosure called hot cell. The quality of air inside the hot cell must be controlled in order to prevent the contamination of the product with particulate material or microorganisms. On the other hand, the hot cell must prevent external contamination with radioactive material. The aim of this work is to discuss the special requirements for hot cells taking in account the national rules for injectable pharmaceutical products and international standards available. Ventilation of radiopharmaceutical production facilities should meet the requirement to prevent the contamination of products and the exposure of working personnel to radioactivity. Positive pressure areas should be used to process sterile products. In general, any radioactivity should handle within specifically designed areas maintained under negative pressures. The production of sterile radioactive products should therefore be carried out under negative pressure surrounded by a positive pressure zone ensuring that appropriate air quality requirements are met. Some of the recent developments in the use of radioisotopes in medical field have also significantly impacted on the evolution of handling facilities. Application of pharmaceutical GMP requirements for air quality and processing conditions in the handling facilities of radioactive pharmaceuticals has led to significant improvements in the construction of isolator-like hot cells and clean rooms with HEPA filtered ventilation and air conditioning (HVAC) systems. Clean grade A (class 100) air quality hot cells are now available commercially, but in a high cost

Air system in the hot cell for injectable radiopharmaceutical production: requirements for personnel and environment safety and protection of the product

International Nuclear Information System (INIS)

Campos, Fabio E.; Araujo, Elaine B.

2009-01-01

Radiopharmaceuticals are applied in Nuclear Medicine in diagnostic and therapeutic procedures and must be manufactured in accordance with the basic principles of Good Manufacturing Practices (GMP) for sterile pharmaceutical products. In order to prevent the uncontrolled spread of radioactive contamination, the processing of radioactive materials requires an exhausted and shielded special enclosure called hot cell. The quality of air inside the hot cell must be controlled in order to prevent the contamination of the product with particulate material or microorganisms. On the other hand, the hot cell must prevent external contamination with radioactive material. The aim of this work is to discuss the special requirements for hot cells taking in account the national rules for injectable pharmaceutical products and international standards available. Ventilation of radiopharmaceutical production facilities should meet the requirement to prevent the contamination of products and the exposure of working personnel to radioactivity. Positive pressure areas should be used to process sterile products. In general, any radioactivity should handle within specifically designed areas maintained under negative pressures. The production of sterile radioactive products should therefore be carried out under negative pressure surrounded by a positive pressure zone ensuring that appropriate air quality requirements are met. Some of the recent developments in the use of radioisotopes in medical field have also significantly impacted on the evolution of handling facilities. Application of pharmaceutical GMP requirements for air quality and processing conditions in the handling facilities of radioactive pharmaceuticals has led to significant improvements in the construction of isolator-like hot cells and clean rooms with HEPA filtered ventilation and air conditioning (HVAC) systems. Clean grade A (class 100) air quality hot cells are now available commercially, but in a high cost

Design of a microbial fuel cell and its transition to microbial electrolytic cell for hydrogen production by electrohydrogenesis.

Science.gov (United States)

Gupta, Pratima; Parkhey, Piyush; Joshi, Komal; Mahilkar, Anjali

2013-10-01

Anaerobic bacteria were isolated from industrial wastewater and soil samples and tested for exoelectrogenic activity by current production in double chambered microbial fuel cell (MFC), which was further transitioned into a single chambered microbial electrolytic cell to test hydrogen production by electrohydrogenesis. Of all the cultures, the isolate from industrial water sample showed the maximum values for current = 0.161 mA, current density = 108.57 mA/m2 and power density = 48.85 mW/m2 with graphite electrode. Maximum voltage across the cell, however, was reported by the isolate from sewage water sample (506 mv) with copper as electrode. Tap water with KMnO4 was the best cathodic electrolyte as the highest values for all the measured MFC parameters were reported with it. Once the exoelectrogenic activity of the isolates was confirmed by current production, these were tested for hydrogen production in a single chambered microbial electrolytic cell (MEC) modified from the MFC. Hydrogen production was reported positive from co-culture of isolates of both the water samples and co-culture of one soil and one water sample. The maximum rate and yield of hydrogen production was 0.18 m3H2/m3/d and 3.2 mol H2/mol glucose respectively with total hydrogen production of 42.4 mL and energy recovery of 57.4%. Cumulative hydrogen production for a five day cycle of MEC operation was 0.16 m3H2/m3/d.

Single Cell Responses to Spatially Controlled Photosensitized Production of Extracellular Singlet Oxygen

DEFF Research Database (Denmark)

Pedersen, Brian Wett; Sinks, Louise E.; Breitenbach, Thomas

2011-01-01

The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served...... to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed post-irradiation “waiting period” before necrosis became apparent depended on (a) the distance...... between the cell membrane and the domain irradiated, (b) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced, and (c) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach...

A platform for high-throughput bioenergy production phenotype characterization in single cells

Science.gov (United States)

Kelbauskas, Laimonas; Glenn, Honor; Anderson, Clifford; Messner, Jacob; Lee, Kristen B.; Song, Ganquan; Houkal, Jeff; Su, Fengyu; Zhang, Liqiang; Tian, Yanqing; Wang, Hong; Bussey, Kimberly; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers. PMID:28349963

Apparatus of hot cell for iodine-123 production

International Nuclear Information System (INIS)

Almeida, G.L. de; Rautenberg, F.A.; Souza, A.S.F. de.

1986-01-01

The hot cell installation at IEN cyclotron (Brazilian-CNEN) for sup(123)I production is presented. Several devices, such as, tube furnace coupling system, tube furnace driving system, sup(123)I target transfer system, product extraction system, furnace control system, and effluent systems, were constructed and modified for implanting process engineering. The requirements of safety engineering for operation process were based on ALARA concept. (M.C.K.)

IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

Science.gov (United States)

Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

2017-08-01

Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

Human umbilical cord mesenchymal stem cells increase interleukin-9 production of CD4+ T cells

OpenAIRE

Yang, Zhou Xin; Chi, Ying; Ji, Yue Ru; Wang, You Wei; Zhang, Jing; Luo, Wei Feng; Li, Li Na; Hu, Cai Dong; Zhuo, Guang Sheng; Wang, Li Fang; Han, Zhi-Bo; Han, Zhong Chao

2017-01-01

Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhance...

Ethanol production by immobilized cells with forced substrate supply

Energy Technology Data Exchange (ETDEWEB)

Mitani, Y.; Nishizawa, Y.; Nagai, S.

1984-01-01

Ethanol fermentation by a forced substrate supply into an immobilized cell layer was carried out to increase the ethanol production rate and to eliminate the diffusion dependency of substrate supply in an ordinary immobilized cell reaction. Saccharomyces cerevisiae IFO 2347 was immobilized in a mixture of k-carrageenan, locust bean gum, and celite (2: 0.5: 40 wt/vol %). A glucose minimal medium was fed into the immobilized cell layer (5 to 22 mm in thickness) at retention times between 0.6 and 2.8 h under pressure. The stable ethanol fermentation could be maintained for more than 3 weeks with an ethanol yield of 0.48 g ethanol/g glucose and ethanol productivity of 63 g.(l gel)/sup -1/.h/sup -1/ at a retention time of 1.5 h. The yeast cells were well distributed through the gel layer with a vertical gradient, and an average cell density was ca. 8.0 X 10/sup 9/ cells/ml gel, 4-fold higher than that of ordinary immobilized cells. A small filter press reactor was constructed to examine the applicability of ethanol fermentation with this forced substrate supply. The operation could be continued for a month at a retention time of 2 h yielding 96 g/l of ethanol from 200 g/l of glucose. 6 references, 5 figures, 3 tables.

Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study).

Science.gov (United States)

Hagendoorn, MJM.; Wagner, A. M.; Segers, G.; Van Der Plas, LHW.; Oostdam, A.; Van Walraven, H. S.

1994-10-01

In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

The role of adrenergic activation on murine luteal cell viability and progesterone production.

Science.gov (United States)

Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

2016-09-15

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

Live Cells Decreased Methane Production in Intestinal Content of Pigs

Directory of Open Access Journals (Sweden)

Y. L. Gong

2013-06-01

Full Text Available An in vitro gas production technique was used in this study to elucidate the effect of two strains of active live yeast on methane (CH4 production in the large intestinal content of pigs to provide an insight to whether active live yeast could suppress CH4 production in the hindgut of pigs. Treatments used in this study include blank (no substrate and no live yeast cells, control (no live yeast cells and yeast (YST supplementation groups (supplemented with live yeast cells, YST1 or YST2. The yeast cultures contained 1.8×1010 cells per g, which were added at the rates of 0.2 mg and 0.4 mg per ml of the fermented inoculum. Large intestinal contents were collected from 2 Duroc×Landrace×Yorkshire pigs, mixed with a phosphate buffer (1:2, and incubated anaerobically at 39°C for 24 h using 500 mg substrate (dry matter (DM basis. Total gas and CH4 production decreased (p<0.05 with supplementation of yeast. The methane production reduction potential (MRP was calculated by assuming net methane concentration for the control as 100%. The MRP of yeast 2 was more than 25%. Compared with the control group, in vitro DM digestibility (IVDMD and total volatile fatty acids (VFA concentration increased (p<0.05 in 0.4 mg/ml YST1 and 0.2 mg/ml YST2 supplementation groups. Proportion of propionate, butyrate and valerate increased (p<0.05, but that of acetate decreased (p<0.05, which led to a decreased (p<0.05 acetate: propionate (A: P ratio in the both YST2 treatments and the 0.4 mg/ml YST 1 supplementation groups. Hydrogen recovery decreased (p<0.05 with yeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (p<0.05 with yeast supplementation after 24 h of incubation. Our results suggest that live yeast cells suppressed in vitro CH4 production when inoculated into the large intestinal contents of pigs and shifted the fermentation pattern to favor propionate production together with an increased population of acetogenic

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies.

Science.gov (United States)

Abbasalizadeh, Saeed; Baharvand, Hossein

2013-12-01

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing. © 2013.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Science.gov (United States)

Yuk, Inn H; Russell, Stephen; Tang, Yun; Hsu, Wei-Ting; Mauger, Jacob B; Aulakh, Rigzen P S; Luo, Jun; Gawlitzek, Martin; Joly, John C

2015-01-01

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes. © 2014 American Institute of Chemical Engineers.

Improving Labor Productivity and Labor Elasticity at Multiproduct Japanese Cuisine Restaurant Introducing Cell-Production System

OpenAIRE

Shimamura , Takeshi; Takenaka , Takeshi; Ohura , Syuichi

2013-01-01

Part III: Sustainable Services; International audience; This study examined improvement of labor productivity and elasticity of labor hour on sales of a multiproduct Japanese cuisine restaurant. Conventionally, multiproduct restaurant operations include a line production system in the kitchen. Japanese chefs are assumed to be low-skilled workers with staff members supported by someone. A cell production system is introduced into a Japanese Cuisine restaurant to improve it. Results show that t...

Müller Glia, Vision-Guided Ocular Growth, Retinal Stem Cells, and a Little Serendipity

Science.gov (United States)

2011-01-01

Hypothesis-driven science is expected to result in a continuum of studies and findings along a discrete path. By comparison, serendipity can lead to new directions that branch into different paths. Herein, I describe a diverse series of findings that were motivated by hypotheses, but driven by serendipity. I summarize how investigations into vision-guided ocular growth in the chick eye led to the identification of glucagonergic amacrine cells as key regulators of ocular elongation. Studies designed to assess the impact of the ablation of different types of neurons on vision-guided ocular growth led to the finding of numerous proliferating cells within damaged retinas. These proliferating cells were Müller glia–derived retinal progenitors with a capacity to produce new neurons. Studies designed to investigate Müller glia–derived progenitors led to the identification of a domain of neural stem cells that form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Accelerated ocular growth, caused by visual deprivation, stimulated the proliferation of CMZ progenitors. We formulated a hypothesis that growth-regulating glucagonergic cells may regulate both overall eye size (scleral growth) and the growth of the retina (proliferation of CMZ cells). Subsequent studies identified unusual types of glucagonergic neurons with terminals that ramify within the CMZ; these cells use visual cues to control equatorial ocular growth and the proliferation of CMZ cells. Finally, while studying the signaling pathways that stimulate CMZ and Müller glia–derived progenitors, serendipity led to the discovery of a novel type of glial cell that is scattered across the inner retinal layers. PMID:21960640

Choosing the right platform for the right product: Sustainable production of chemicals in microbial cell factories

DEFF Research Database (Denmark)

Herrgard, Markus

The Novo Nordisk Foundation Center for Biosustainability (CFB) is a new non-profit research center focused on sustainable production of biochemicals and therapeutic proteins using microbial and mammalian cell factories. The work at CFB is organized around an iterative loop where cell factories...

Bioreactors for high cell density and continuous multi-stage cultivations: options for process intensification in cell culture-based viral vaccine production.

Science.gov (United States)

Tapia, Felipe; Vázquez-Ramírez, Daniel; Genzel, Yvonne; Reichl, Udo

2016-03-01

With an increasing demand for efficacious, safe, and affordable vaccines for human and animal use, process intensification in cell culture-based viral vaccine production demands advanced process strategies to overcome the limitations of conventional batch cultivations. However, the use of fed-batch, perfusion, or continuous modes to drive processes at high cell density (HCD) and overextended operating times has so far been little explored in large-scale viral vaccine manufacturing. Also, possible reductions in cell-specific virus yields for HCD cultivations have been reported frequently. Taking into account that vaccine production is one of the most heavily regulated industries in the pharmaceutical sector with tough margins to meet, it is understandable that process intensification is being considered by both academia and industry as a next step toward more efficient viral vaccine production processes only recently. Compared to conventional batch processes, fed-batch and perfusion strategies could result in ten to a hundred times higher product yields. Both cultivation strategies can be implemented to achieve cell concentrations exceeding 10(7) cells/mL or even 10(8) cells/mL, while keeping low levels of metabolites that potentially inhibit cell growth and virus replication. The trend towards HCD processes is supported by development of GMP-compliant cultivation platforms, i.e., acoustic settlers, hollow fiber bioreactors, and hollow fiber-based perfusion systems including tangential flow filtration (TFF) or alternating tangential flow (ATF) technologies. In this review, these process modes are discussed in detail and compared with conventional batch processes based on productivity indicators such as space-time yield, cell concentration, and product titers. In addition, options for the production of viral vaccines in continuous multi-stage bioreactors such as two- and three-stage systems are addressed. While such systems have shown similar virus titers compared to

Work productivity in rhinitis using cell phones

DEFF Research Database (Denmark)

Bousquet, J.; Bewick, Bridgette M; Arnavielhe, S

2017-01-01

Allergic rhinitis often impairs social life and performance. The aim of this cross-sectional study was to use cell phone data to assess the impact on work productivity of uncontrolled rhinitis assessed by visual analogue scale (VAS). A mobile phone app (Allergy Diary, Google Play Store and Apple ...

Cell immobilization for production of lactic acid biofilms do it naturally.

Science.gov (United States)

Dagher, Suzanne F; Ragout, Alicia L; Siñeriz, Faustino; Bruno-Bárcena, José M

2010-01-01

Interest in natural cell immobilization or biofilms for lactic acid fermentation has developed considerably over the last few decades. Many studies report the benefits associated with biofilms as industrial methods for food production and for wastewater treatment, since the formation represents a protective means of microbial growth offering survival advantages to cells in toxic environments. The formation of biofilms is a natural process in which microbial cells adsorb to a support without chemicals or polymers that entrap the cells and is dependent on the reactor environment, microorganism, and characteristics of the support. These unique characteristics enable biofilms to cause chronic infections, disease, food spoilage, and devastating effects as in microbial corrosion. Their distinct resistance to toxicity, high biomass potential, and improved stability over cells in suspension make biofilms a good tool for improving the industrial economics of biological lactic acid production. Lactic acid bacteria and specific filamentous fungi are the main sources of biological lactic acid. Over the past two decades, studies have focused on improving the lactic acid volumetric productivity through reactor design development, new support materials, and improvements in microbial production strains. To illustrate the operational designs applied to the natural immobilization of lactic acid producing microorganisms, this chapter presents the results of a search for optimum parameters and how they are affected by the physical, chemical, and biological variables of the process. We will place particular emphasis upon the relationship between lactic acid productivity attained by various types of reactors, supports, media formulations, and lactic acid producing microorganisms. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Industrial production of clotting factors: Challenges of expression, and choice of host cells.

Science.gov (United States)

Kumar, Sampath R

2015-07-01

The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

First complete and productive cell culture model for members of the genus Iridovirus.

Science.gov (United States)

D'Costa, Susan M; Vigerust, David J; Perales-Hull, Marsha R; Lodhi, Sundus A; Viravathana, Polrit; Bilimoria, Shän L

2012-11-01

Chilo iridescent virus (CIV; the type strain of the genus Iridovirus) replicates productively in larvae of the boll weevil, Anthonomus grandis. This study focuses on characterizing productive infections of a boll weevil cell line, BRL-AG-3A (AG3A), starting with CIV reared in the waxworm, Galleria mellonella. We show that CIV can be continually and productively passaged to high titer in AG3A cells. The replication of larval-derived CIV in AG3A was analyzed by observing viral DNA replication and restriction endonuclease digestion profiles, morphogenesis, and infectivity using TCID(50) assays with AG3A as an indicator cell line. The data showed that virus passaged in the AG3A host is stable. AG3A cells are more efficient than previously utilized CF-124T cells from Choristoneura fumiferana. This system constitutes a superior model for cellular and molecular studies on CIV; it represents the first complete, productive cell culture model for the replication of CIV or any member of the genus Iridovirus.

Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Huang, Li-Wen [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hsieh, Bau-Shan; Cheng, Hsiao-Ling [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hu, Yu-Chen [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chang, Wen-Tsan [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Division of Hepatobiliarypancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan (China); Chang, Kee-Lung, E-mail: Chang.KeeLung@msa.hinet.net [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

2012-01-15

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M

Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. Characterization of a soluble suppressor of B cell immunoglobulin production

International Nuclear Information System (INIS)

Fleisher, T.A.; Greene, W.C.; Blaese, R.M.; Waldmann, T.A.

1981-01-01

Human peripheral blood mononuclear cells (PBMC) activated with the mitogenic lectin concanavalin A (Con A) elaborate a soluble immune suppressor supernatant (SISS) that contains at least 2 distinct suppressor factors. One of these, SISS-B, inhibits polyclonal B cell immunoglobulin production, whereas the other, SISS-T, suppresses T cell proliferation to both mitogens and antigens. The latter mediator is discussed in the companion paper. Characteristics of the human soluble suppressor of B cell immunoglobulin production (SISS-B) include: 1) inhibition by a noncytotoxic mechanism, 2) loss of activity in the presence of the monosaccharide L-rhamnose, 3) appearance within 8 to 16 hr after the addition of Con A, 4) elaboration by cells irradiated with 500 or 2000 rads, 5) production by highly purified T cells, 6) stability at pH 2.5 but instability at 56/sup o/C, and 7) m.w. of 60 to 80,000. These data indicate that after Con A activation, selected T cells not only become potent suppressor cells, but also generate a soluble saccharide-specific factor(s) that inhibits polyclonal immunoglobulin production by human B cells

Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells

International Nuclear Information System (INIS)

Kang, Wen; Mukerjee, Ruma; Gartner, Jared J.; Hatzigeorgiou, Artemis G.; Sandri-Goldin, Rozanne M.; Fraser, Nigel W.

2006-01-01

The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells

Development, Qualification and Production of Space Solar Cells with 30% EOL Efficiency

Science.gov (United States)

Guter, Wolfgang; Ebel, Lars; Fuhrmann, Daniel; Kostler, Wolfgang; Meusel, Matthias

2014-08-01

AZUR SPACE's latest qualified solar cell product 3G30-advanced provides a high end-of-life (EOL) efficiency of 27.8% for 5E14 (1 MeV e-/cm2) at low production costs. In order to further reduce the mass, the 3G30-advanced was thinned down to as thin as 20 μm and tested in space. Next generation solar cells must exceed the EOL efficiency of the 3G30-advanced and therefore will utilize the excess current of the Ge subcell. This can be achieved by a metamorphic cell concept. While average beginning-of-life efficiencies above 31% have already been demonstrated with upright metamorphic triple-junction cells, AZUR's next generation product will comprise a metamorphic 4- junction device targeting 30% EOL.

Adaptive Regulation of Osteopontin Production by Dendritic Cells Through the Bidirectional Interaction With Mesenchymal Stromal Cells

Directory of Open Access Journals (Sweden)

Sara Scutera

2018-06-01

Full Text Available Mesenchymal stromal cells (MSCs exert immunosuppressive effects on immune cells including dendritic cells (DCs. However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN, a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β. OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29. Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2. Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+ are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with

Durability of solid oxide electrolysis cells for hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Hauch, A.; Hoejgaard Jensen, S.; Dalgaard Ebbesen, S.

2007-05-15

In the perspective of the increasing interest in renewable energy and hydrogen economy, the reversible solid oxide cells (SOCs) is a promising technology as it has the potential of providing efficient and cost effective hydrogen production by high temperature electrolysis of steam (HTES). Furthermore development of such electrolysis cells can gain from the results obtained within the R and D of SOFCs. For solid oxide electrolysis cells (SOEC) to become interesting from a technological point of view, cells that are reproducible, high performing and long-term stable need to be developed. In this paper we address some of the perspectives of the SOEC technology i.e. issues such as a potential H2 production price as low as 0.71 US dollar/kg H{sub 2} using SOECs for HTES; is there a possible market for the electrolysers? and what R and D steps are needed for the realisation of the SOEC technology? In the experimental part we present electrolysis test results on SOCs that have been optimized for fuel cell operation but applied for HTES. The SOCs are produced on a pre-pilot scale at Risoe National Laboratory. These cells have been shown to have excellent initial electrolysis performance, but the durability of such electrolysis cells are not optimal and examples of results from SOEC tests over several hundreds of hours are given here. The long-term tests have been run at current densities of -0.5 A/cm{sup 2} and -1 A/cm{sup 2}, temperatures of 850 deg. C and 950 deg. C and p(H{sub 2}O)/p(H{sub 2}) of 0.5/0.5 and 0.9/0.1. Long-term degradation rates are shown to be up to 5 times higher for SOECs compared to similar SOFC testing. Furthermore, hydrogen and synthetic fuel production prices are calculated using the experimental results from long-term electrolysis test as input and a short outlook for the future work on SOECs will be given as well. (au)

Optimized microbial cells for production of melatonin and other compounds

DEFF Research Database (Denmark)

2017-01-01

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using...

Production of soluble Neprilysin by endothelial cells

International Nuclear Information System (INIS)

Kuruppu, Sanjaya; Rajapakse, Niwanthi W.; Minond, Dmitriy; Smith, A. Ian

2014-01-01

Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC 50 values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17

DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro

DEFF Research Database (Denmark)

Müller, Hanna; Weiss, Christel; Renner, Marcus

2017-01-01

Meconium aspiration syndrome (MAS) is characterized by surfactant inactivation and inflammation. As lung epithelial cells up-regulate nitric oxide (NO) in response to inflammation, the NO production following meconium exposition was examined in relation to expression of Deleted in Malignant Brain...... NO production than the DMBT1- cells (p = 0.0090). Meconium led in DMBT1- and DMBT1+ cells to elevated NO levels (p production in DMBT1+ cells (p = 0.0476), but NO levels remained above...... NO production from DMBT1- cells (p = 0.0289). Dexamethasone diminished NO production in DMBT1+ cells after meconium exposition (p = 0.0076). Combined addition of LPS and meconium significantly increased NO production in both cell types (p

CD4+ T cell-derived novel peptide Thp5 induces interleukin-4 production in CD4+ T cells to direct T helper 2 cell differentiation.

Science.gov (United States)

Khan, Mohd Moin; Chatterjee, Samit; Dwivedi, Ved Prakash; Pandey, Nishant Kumar; Singh, Yogesh; Tousif, Sultan; Bhavesh, Neel Sarovar; Van Kaer, Luc; Das, Jyoti; Das, Gobardhan

2012-01-20

The differentiation of naïve CD4(+) T cells into T helper 2 (Th2) cells requires production of the cytokine IL-4 in the local microenvironment. It is evident that naïve/quiescently activated CD4(+) T cells produce the IL-4 that drives Th2 cell differentiation. Because early production of IL-4 in naïve T cells leads to preferential Th2 cell differentiation, this process needs to be tightly regulated so as to avoid catastrophic and misdirected Th2 cell differentiation. Here, we show that Thp5, a novel peptide with structural similarity to vasoactive intestinal peptide, regulates production of early IL-4 in newly activated CD4(+) T cells. Induction of IL-4 in CD4(+) T cells by Thp5 is independent of the transcription factor STAT6 but dependent on ERK1/2 signaling. Furthermore, cytokines (IL-12 and TGF-β) that promote the differentiation of Th1 or Th17 cells inhibit Thp5 induction, thus suppressing Th2 cell differentiation. We further showed that Thp5 enhances Th2 responses and exacerbates allergic airway inflammation in mice. Taken together, our findings reveal that early activated CD4(+) T cells produce Thp5, which plays a critical role as a molecular switch in the differentiation of Th cells, biasing the response toward the Th2 cell phenotype.

Benfotiamine prevents increased β-amyloid production in HEK cells induced by high glucose.

Science.gov (United States)

Sun, Xiao-Jing; Zhao, Lei; Zhao, Na; Pan, Xiao-Li; Fei, Guo-Qiang; Jin, Li-Rong; Zhong, Chun-Jiu

2012-10-01

To determine whether high glucose enhances β-amyloid (Aβ) production in HEK293 Swedish mutant (APPsw) cells with Aβ precursor protein (APP) overexpression, and whether under this condition benfotiamine reduces the increased Aβ production. HEK293 APPsw cells were cultured with different concentrations of glucose for different times. The Aβ content in the supernatant was determined by ELISA. To investigate the mechanism by which benfotiamine reduced Aβ production, glycogen synthase kinase-3 (GSK-3) activity and expression were measured after the cells were cultured with 5.5 g/L glucose for 12 h. With 1.0, 3.0, 4.5, 5.5, 6.5, 7.5, 8.5, or 10.5 g/L glucose, Aβ production by HEK293 APPsw cells was highest in the presence of 5.5 g/L glucose for 6 and 12 h. The difference in Aβ content between 5.5 and 1.0 g/L was most marked after incubation for 12 h. Benfotiamine at 20 and 40 μg/mL significantly reduced Aβ production in cells incubated with 5.5 g/L glucose for 12 h. Moreover, 40 μg/mL benfotiamine significantly enhanced the ratio of phosphorylated GSK-3 to total GSK-3, together with consistent down-regulation of GSK-3 activity. High glucose increases Aβ production by HEK293 APPsw cells while benfotiamine prevents this increase. This is correlated with the modulation of GSK-3 activity.

Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells.

Science.gov (United States)

Yamashita, Tomoki; Takayama, Kazuo; Sakurai, Fuminori; Mizuguchi, Hiroyuki

2018-02-19

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10 9  cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Production cell implementation in the facade execution: a construction case study

Directory of Open Access Journals (Sweden)

Renato Nunes Mariz

2014-05-01

Full Text Available The construction industry is an environment with much waste. An alternative that could minimize this problem is Lean Thinking, as it focuses on eliminating waste. A widely used tool in lean manufacturing is the production cell, whose main goal is to keep production in continuo flow. In the construction context, the applications of this tool are still incipient, arousing the interest to be further investigated. The aim of this study is to analyze how a production cell was applied in a facade service execution and evaluate this result. We used the case study method in a contractor with experience in facades execution. We collected qualitative data (in situ observations and interviews with employees and quantitative (time and cost data supplied by the contractor. After the data were analyzed (before and after cell implementation Through analysis and literature review we decided to follow those steps: a methods b materials c people d equipments and machines. The results point out improvement in the employee relationship with the administration, increased employee commitment, cost savings with direct labor and increase the pay received by employees. This paper contributes to expanding the use of this tool (production cell in the construction and shows that through this application, many benefits can be achieved.

MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

Energy Technology Data Exchange (ETDEWEB)

H.C. Maru; M. Farooque

2005-03-01

The program was designed to advance the carbonate fuel cell technology from full-size proof-of-concept field test to the commercial design. DOE has been funding Direct FuelCell{reg_sign} (DFC{reg_sign}) development at FuelCell Energy, Inc. (FCE, formerly Energy Research Corporation) from an early state of development for stationary power plant applications. The current program efforts were focused on technology and system development, and cost reduction, leading to commercial design development and prototype system field trials. FCE, in Danbury, CT, is a world-recognized leader for the development and commercialization of high efficiency fuel cells that can generate clean electricity at power stations, or at distributed locations near the customers such as hospitals, schools, universities, hotels and other commercial and industrial applications. FCE has designed three different fuel cell power plant models (DFC300A, DFC1500 and DFC3000). FCE's power plants are based on its patented DFC{reg_sign} technology, where a hydrocarbon fuel is directly fed to the fuel cell and hydrogen is generated internally. These power plants offer significant advantages compared to the existing power generation technologies--higher fuel efficiency, significantly lower emissions, quieter operation, flexible siting and permitting requirements, scalability and potentially lower operating costs. Also, the exhaust heat by-product can be used for cogeneration applications such as high-pressure steam, district heating and air conditioning. Several sub-MW power plants based on the DFC design are currently operating in Europe, Japan and the US. Several one-megawatt power plant design was verified by operation on natural gas at FCE. This plant is currently installed at a customer site in King County, WA under another US government program and is currently in operation. Because hydrogen is generated directly within the fuel cell module from readily available fuels such as natural gas and

Whey utilization for single-cell protein production

Energy Technology Data Exchange (ETDEWEB)

Barraquio, V; Silverio, L G; Revilleza, R P; Fernadez, W L

1980-01-01

The production of single-cell protein by yeast assimilation of lactose in soft cheese whey was studied using Candida pseudotropicalis as a test organism. Under shake-flask cultivation conditions with deproteinized whey as the medium, lactose (initially 4.20%) was completely assimilated in 48h; cell mass was 5.56 mg/mL after 72h; and average protein content of the dried mass was approximately 11.8%. Batch cultivation using undeproteinized whey resulted in a faster lactose utilization rate from an initial 3.93% to a residual 0.56% in 12 h; cell mass was 8.41 mg/mL in 10 h; and average protein was approximately 37.7%. In a semicontinuous culture with 10 to the power of 7 viable cells/mL as initial cell concentration, 15.69 mg/mL cell mass with a mean protein content of approximately 21.4% could be produced and lactose could be considerably consumed (from an initial 4.75% to a residual 0.42%) within 13-14 h. Supplementation with (NH/sub 4/)/sub 2/S0/sub 4/ and KH/sub 2/P0/sub 4/ did not increase cell mass (12.47 mg/mL in 12 h) and hasten lactose assimulation (from initial 4.49% to residual 0.3% in 12 h). Average protein content was approximately 31%. Cell mass yield was established as 0.29 mg yeast cell/mg lactose consumed. Factors that might have affected protein content are also discussed.

Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

DEFF Research Database (Denmark)

Zhou, Yongjin J.; Buijs, Nicolaas A; Zhu, Zhiwei

2016-01-01

Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level p...

Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

International Nuclear Information System (INIS)

Kolokoltsova, Olga A.; Domina, Aaron M.; Kolokoltsov, Andrey A.; Davey, Robert A.; Weaver, Scott C.; Watowich, Stanley J.

2008-01-01

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection

Fenofibrate induces ketone body production in melanoma and glioblastoma cells

Directory of Open Access Journals (Sweden)

Maja M Grabacka

2016-02-01

Full Text Available Ketone bodies (beta-hydroxybutyrate, bHB, acetoacetate are mainly produced in the liver during prolonged fasting or starvation. bHB is a very efficient energy substrate for sustaining ATP production in peripheral tissues; importantly its consumption is preferred over glucose. However, the majority of malignant cells, particularly cancer cells of neuroectodermal origin such as glioblastoma, are not able to use ketone bodies as a source of energy. Here, we report a novel observation that fenofibrate, a synthetic peroxisome proliferator-activated receptor alpha (PPARa agonist, induces bHB production in melanoma and glioblastoma cells, as well as in neurospheres composed of nontransformed cells. Unexpectedly, this effect is not dependent on PPARa activity or its expression level. The fenofibrate-induced ketogenesis is accompanied by growth arrest and down-regulation of transketolase, but the NADP/NADPH and GSH/GSSG ratios remain unaffected. Our results reveal a new, intriguing aspect of cancer cell biology and highlight the benefits of fenofibrate as a supplement to both canonical and dietary (ketogenic therapeutic approaches against glioblastoma.

Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC Damages.

Directory of Open Access Journals (Sweden)

Yumei Zhang

Full Text Available Here, we studied the underlying mechanism of aldosterone (Aldo-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L inhibited human umbilical vein endothelial cells (HUVEC survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18 production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P, an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS inhibitor PDMP or the ceramide (C6 potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1 is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.

Electrocatalysis research for fuel cells and hydrogen production

CSIR Research Space (South Africa)

Mathe, MK

2012-01-01

Full Text Available The CSIR undertakes research in the Electrocatalysis of fuel cells and for hydrogen production. The Hydrogen South Africa (HySA) strategy supports research on electrocatalysts due to their importance to the national beneficiation strategy. The work...

Concise Review: Bone Marrow Mononuclear Cells for the Treatment of Ischemic Syndromes: Medicinal Product or Cell Transplantation?

Science.gov (United States)

Rico, Laura; Herrera, Concha

2012-01-01

In November of 2011, the Committee for Advanced Therapies (CAT) of the European Medicines Agency (EMA) published two scientific recommendations regarding the classification of autologous bone marrow-derived mononuclear cells (BM-MNCs) and autologous bone marrow-derived CD133+ cells as advanced therapy medicinal products (ATMPs), specifically tissue-engineered products, when intended for regeneration in ischemic heart tissue on the basis that they are not used for the same essential function (hematological restoration) that they fulfill in the donor. In vitro and in vivo evidence demonstrates that bone marrow cells are physiologically involved in adult neovascularization and tissue repair, making their therapeutic use for these purposes a simple exploitation of their own essential functions. Therefore, from a scientific/legal point of view, nonsubstantially manipulated BM-MNCs and CD133+ cells are not an ATMP, because they have a physiological role in the processes of postnatal neovascularization and, when used therapeutically for vascular restoration in ischemic tissues, they are carrying out one of their essential physiological functions (the legal definition recognizes that cells can have several essential functions). The consequences of classifying BM-MNCs and CD133+ cells as medicinal products instead of cellular transplantation, like bone marrow transplantation, in terms of costs and time for these products to be introduced into clinical practice, make this an issue of crucial importance. Therefore, the recommendations of EMA/CAT could be reviewed in collaboration with scientific societies, in light of organizational and economic consequences as well as scientific knowledge recently acquired about the mechanisms of postnatal neovascularization and the function of bone marrow in the regeneration of remote tissues. PMID:23197819

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

Science.gov (United States)

Lee, Byung-Chul; Kim, Hyung-Sik; Shin, Tae-Hoon; Kang, Insung; Lee, Jin Young; Kim, Jae-Jun; Kang, Hyun Kyoung; Seo, Yoojin; Lee, Seunghee; Yu, Kyung-Rok; Choi, Soon Won; Kang, Kyung-Sun

2016-05-27

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.

Sesamin Ameliorates Advanced Glycation End Products-Induced Pancreatic β-Cell Dysfunction and Apoptosis

Directory of Open Access Journals (Sweden)

Xiang Kong

2015-06-01

Full Text Available Advanced glycation end products (AGEs, the direct modulators of β-cells, have been shown to cause insulin-producing β-cell dysfunction and apoptosis through increase of intracellular reactive oxygen species (ROS production. Sesamin has been demonstrated to possess antioxidative activity. This study was designed to investigate whether sesamin protects against AGEs-evoked β-cell damage via its antioxidant property. The effects of sesamin were examined in C57BL/6J mice and MIN6 cell line. In in vivo studies, mice were intraperitoneally injected with AGEs (120 mg/kg and orally treated with sesamin (160 mg/kg for four weeks. Intraperitoneal glucose tolerance and insulin releasing tests were performed. Insulin content, ROS generation and β-cell apoptosis in pancreatic islets were also measured. In in vitro studies, MIN6 cells were pretreated with sesamin (50 or 100 μM and then exposed to AGEs (200 mg/L for 24 h. Insulin secretion, β-cell death, ROS production as well as expression and activity of NADPH oxidase were determined. Sesamin treatment obviously ameliorated AGE-induced β-cell dysfunction and apoptosis both in vivo and in vitro. These effects were associated with decreased ROS production, down-regulated expression of p67phox and p22phox, and reduced NADPH oxidase activity. These results suggest that sesamin protects β-cells from damage caused by AGEs through suppressing NADPH oxidase-mediated oxidative stress.

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

International Nuclear Information System (INIS)

Shimizu, Takashi; Hirai, Yuko; Murayama, Chiaki; Miyamoto, Akio; Miyazaki, Hitoshi; Miyazaki, Koyomi

2011-01-01

Highlights: → Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression. →Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom. → Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. →Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. → The expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. -- Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

Energy Technology Data Exchange (ETDEWEB)

Shimizu, Takashi, E-mail: shimizut@obihiro.ac.jp [Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan); Hirai, Yuko; Murayama, Chiaki; Miyamoto, Akio [Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan); Miyazaki, Hitoshi [Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Miyazaki, Koyomi [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566 (Japan)

2011-08-19

Highlights: {yields} Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression. {yields}Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom. {yields} Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. {yields}Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. {yields} The expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. -- Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.

Examining the sources of variability in cell culture media used for biopharmaceutical production.

Science.gov (United States)

McGillicuddy, Nicola; Floris, Patrick; Albrecht, Simone; Bones, Jonathan

2018-01-01

Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.

Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

Science.gov (United States)

Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

2014-05-01

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Large Scale Production of Stem Cells and Their Derivatives

Science.gov (United States)

Zweigerdt, Robert

Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

Biphasic effect of oxygen radicals on prostaglandin production by rat mesangial cells

International Nuclear Information System (INIS)

Adler, S.; Stahl, R.A.K.; Baker, P.J.; Chen, Y.P.; Pritzl, P.M.; Couser, W.G.

1987-01-01

Cultured rat mesangial cells were exposed to a reactive oxygen species (ROS) generating system (xanthine plus xanthine oxidase) to explore the effect of ROS on their metabolism of arachidonic acid (AA). Cell viability, as assessed by 51 Cr release, was not affected by the concentrations of xanthine plus xanthine oxidase used. Prostaglandin E 2 (PGE 2 ) production following exposure to increasing quantities of xanthine plus xanthine oxidase was significantly decreased when cells were stimulated with the calcium ionophore A23187 or AA. Maximum suppression of production was seen within 10 min of ROS exposure. Thromboxane B 2 production was similarly decreased. This effect was reversed by addition of catalase to the ROS generating system but not by superoxide dismutase or mannitol, which suggested that H 2 O 2 was the responsible metabolite. High levels of H 2 O 2 suppressed PGE 2 production. Lower levels of H 2 O 2 resulted in significant stimulation of base-line PGE 2 production. Analysis of release of 3 H]AA-labeled metabolites from A23187-stimulated cells showed no effect of H 2 O 2 on phospholipase activity. Thus ROS can stimulate or inhibitor AA metabolism in the glomerular mesangium, which may have important effects on glomerular hemodynamics during glomerular injury

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

Science.gov (United States)

Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

2018-01-01

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

EH AND S ANALYSIS OF DYE-SENSITIZED PHOTOVOLTAIC SOLAR CELL PRODUCTION

International Nuclear Information System (INIS)

BOWERMAN, B.; FTHENAKIS, V.

2001-01-01

Photovoltaic solar cells based on a dye-sensitized nanocrystalline titanium dioxide photoelectrode have been researched and reported since the early 1990's. Commercial production of dye-sensitized photovoltaic solar cells has recently been reported in Australia. In this report, current manufacturing methods are described, and estimates are made of annual chemical use and emissions during production. Environmental, health and safety considerations for handling these materials are discussed. This preliminary EH and S evaluation of dye-sensitized titanium dioxide solar cells indicates that some precautions will be necessary to mitigate hazards that could result in worker exposure. Additional information required for a more complete assessment is identified

Life-cycle analysis of product integrated polymer solar cells

DEFF Research Database (Denmark)

Espinosa Martinez, Nieves; García-Valverde, Rafael; Krebs, Frederik C

2011-01-01

A life cycle analysis (LCA) on a product integrated polymer solar module is carried out in this study. These assessments are well-known to be useful in developmental stages of a product in order to identify the bottlenecks for the up-scaling in its production phase for several aspects spanning from...... economics through design to functionality. An LCA study was performed to quantify the energy use and greenhouse gas (GHG) emissions from electricity use in the manufacture of a light-weight lamp based on a plastic foil, a lithium-polymer battery, a polymer solar cell, printed circuitry, blocking diode......, switch and a white light emitting semiconductor diode. The polymer solar cell employed in this prototype presents a power conversion efficiency in the range of 2 to 3% yielding energy payback times (EPBT) in the range of 1.3–2 years. Based on this it is worthwhile to undertake a life-cycle study...

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

Science.gov (United States)

Davari, Maliheh; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

2013-01-01

Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

Entropy production in a cell and reversal of entropy flow as an anticancer therapy

Institute of Scientific and Technical Information of China (English)

Liao-fu LUO

2009-01-01

The entropy production rate of cancer cells is always higher than healthy cells in the case where no external field is applied. Different entropy production between two kinds of cells determines the direction of entropy flow among cells. The entropy flow is the carrier of information flow. The entropy flow from cancerous cells to healthy cells takes along the harmful information of cancerous cells, propagating its toxic action to healthy tissues. We demonstrate that a low-frequency and low- intensity electromagnetic field or ultrasound irradiation may increase the entropy production rate of a cell in normal tissue than that in cancer and consequently re- verse the direction of entropy current between two kinds of cells. The modification of the PH value of cells may also cause the reversal of the direction of entropy flow between healthy and cancerous cells. Therefore, the bio- logical tissue under the irradiation of an electromagnetic field or ultrasound or under the appropriate change of cell acidity can avoid the propagation of harmful infor- marion from cancer cells. We suggest that this entropy mechanism possibly provides a basis for a novel approach to anticancer therapy.

Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

Science.gov (United States)

Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

2015-07-14

Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

High cell density strategy for poly(3-hydroxybutyrate production by Cupriavidus necator

Directory of Open Access Journals (Sweden)

J. L. Ienczak

2011-12-01

Full Text Available Poly(3-hydroxybutyrate (P(3HB is a carbon and intracellular storage source for different microorganisms and its production can achieve high productivities by means of high cell density cultures. The aim of this study was to propose a high cell density strategy for P(3HB production by Cupriavidus necator. The exponential growth phase demands an accurate control of the oxygen transfer system in the bioreactor, due to maximum specific growth rate (µXr, and, consequently, a maximum specific oxygen uptake rate (QO2, in addition to significant residual biomass (Xr growth in high cell density cultures. In this context, this work investigated the strategy for obtaining high cell density, with the inclusion of a linear growth phase for P(3HB production by C. necator in a fed-batch culture. The linear growth phase was included between the exponential growth phase and the P(3HB production phase as a strategy to reduce the specific growth rate (µXr and specific oxygen uptake rate (QO2, with constant residual biomass growth rate (d(V.Xr/dt = k = constant and linear increase of biomass. Three strategies of culture were performed. The results showed that a high residual biomass concentration (30 gXr.L-1 can be reached by the inclusion of the linear growth strategy and specific growth rates (µXr between 0.08 and 0.05 h-1, at the beginning of the production phase, are necessary to attain a high P(3HB productivity.

Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers

Directory of Open Access Journals (Sweden)

Phoowit Bangrak

2011-06-01

Full Text Available A culture of Saccharomyces cerevisiae M30 entrapped in loofa-reinforced alginate was used for continuous ethanol fermentation in a packed-bed reactor with initial sugar concentrations of 200-248 g/L. Maximum ethanol productivity of 11.5 g/(L·h was obtained at an ethanol concentration of 57.4 g/L, an initial sugar concentration of 220 g/L and a dilution rate (D of 0.2 h-1. However, a maximum ethanol concentration of 82.1 g/L (productivity of 9.0 g/(L·h was obtained at a D of 0.11 h-1. Ethanol productivity in the continuous culture was 6-8-fold higher than that in the batch culture. Due to the developed carrier's high biocompatibility, high porosity, and good mechanical strength, advantages such as cell regeneration, reusability, altered mechanical strength, and high capacity to trap active cells in the reactor were achieved in this study. The immobilized cell reactor was successfully operated for 30 days without any loss in ethanol productivity. The average conversion yield was 0.43-0.45 throughout the entire operation, with an immobilization yield of 47.5%. The final total cell concentration in the reactor was 37.3 g/L (17.7 g/L immobilized cells and 19.6 g/L suspended cells. The concentration of suspended cells in the effluent was 0.8 g/L.

Durable solid oxide electrolysis cells for hydrogen production

DEFF Research Database (Denmark)

Sun, Xiufu; Chen, Ming; Hendriksen, Peter Vang

2014-01-01

production is required for promoting commercialization of the SOEC technology. In this work, we report a recent 4400 hours test of a state-of-the-art Ni-YSZ electrode supported SOEC cell. The cell consists of a Ni-YSZ (YSZ: yttria stabilized zirconia) support and active fuel electrode, an YSZ electrolyte...... that except for the first 250 hours fast initial degradation, for the rest of the testing period, the cell showed rather stable performance with an moderate degradation rate of around 25 mV/1000 h. The electrochemical impedance spectra show that both serial resistance and polarization resistance of the cell...... and changing of porosity inside the active layer. The degree of these microstructural changes becomes less and less severe along the hydrogen-steam flow path. The present test results show that this type of cell can be used for early demonstration electrolysis at 1A/cm2. Future work should be focus on reducing...

Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures.

Science.gov (United States)

Appelhagen, Ingo; Wulff-Vester, Anders Keim; Wendell, Micael; Hvoslef-Eide, Anne-Kathrine; Russell, Julia; Oertel, Anne; Martens, Stefan; Mock, Hans-Peter; Martin, Cathie; Matros, Andrea

2018-06-08

Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes. We have used tobacco lines which constitutively produce high levels of cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside or a novel anthocyanin, acylated cyanidin 3-O-(coumaroyl) rutinoside to generate cell suspension cultures. The cell lines are stable in their production rates and superior to conventional plant cell cultures. Scale-up of anthocyanin production in small scale fermenters has been demonstrated. The cell cultures have also proven to be a suitable system for production of 13 C-labelled anthocyanins. Our method for anthocyanin production is transferable to other plant species, such as Arabidopsis thaliana, demonstrating the potential of this approach for making a wide range of highly-decorated anthocyanins. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

[Regulatory requirements regarding cell-based medicinal products for human and veterinary use - a comparison].

Science.gov (United States)

Kuhlmann-Gottke, Johanna; Duchow, Karin

2015-11-01

At present, there is no separate regulatory framework for cell-based medicinal products (CBMP) for veterinary use at the European or German level. Current European and national regulations exclusively apply to the corresponding medicinal products for human use. An increasing number of requests for the regulatory classification of CBMP for veterinary use, such as allogeneic stem cell preparations and dendritic cell-based autologous tumour vaccines, and a rise in scientific advice for companies developing these products, illustrate the need for adequate legislation. Currently, advice is given and decisions are made on a case-by-case basis regarding the regulatory classification and authorisation requirements.Since some of the CBMP - in particular in the area of stem-cell products - are developed in parallel for human and veterinary use, there is an urgent need to create specific legal definitions, regulations, and guidelines for these complex innovative products in the veterinary sector as well. Otherwise, there is a risk that that the current legal grey area regarding veterinary medicinal products will impede therapeutic innovations in the long run. A harmonised EU-wide approach is desirable. Currently the European legislation on veterinary medicinal products is under revision. In this context, veterinary therapeutics based on allogeneic cells and tissues will be defined and regulated. Certainly, the legal framework does not have to be as comprehensive as for human CBMP; a leaner solution is conceivable, similar to the special provisions for advanced-therapy medicinal products laid down in the German Medicines Act.

T cell subsets related with a sex difference in IL-5 production.

Science.gov (United States)

Okuyama, Kaori; Hamanaka, Yuka; Kawano, Tasuku; Ohkawara, Yuichi; Takayanagi, Motoaki; Kikuchi, Toshiaki; Ohno, Isao

2011-01-01

Before puberty, the prevalence and severity of asthma are higher in boys than in girls, but this pattern is reversed after puberty. The underlying mechanisms of these gender differences in asthma are not fully understood. Using murine models of allergic asthma, a sex difference in Th2 cytokine production has been suggested to contribute to the gender differences in asthma. Therefore, we determined which subsets of T cells are involved in the sex difference in Th2 cytokine production. Splenocytes from wild-type mice and CD4+ T cell-, CD8+ T cell-, and iNKT cell-deficient mice were stimulated with anti-CD3/CD28 antibodies for 3 days, and the concentrations of IL-4, IL-5, IL-13, and IFN-γ in the cultures were measured by ELISA. IL-5, but not IL-4 and IL-13, concentrations in culture derived from female wild-type mice were significantly higher than those in male wild-type mice. The sex difference in IL-5 concentrations was not observed in the cultures of splenocytes from CD4+ and CD8+ T cell-deficient mice. The disappearance of the sex differences in CD4+ and CD8+ T cell-deficient mice was attributable to a decrease in IL-5 concentration in female mice and an increase in IL-5 concentration in male mice. In iNKT cell-deficient mice, the sex difference was still observed. There was no significant difference between the sexes in any type of mice with respect to IFN-γ production. There was a sex difference in IL-5 production by splenocytes stimulated by TCR activation. The difference might be attributable to sex differences in CD4+ and CD8+ T cell functions. Copyright © 2011 S. Karger AG, Basel.

Collectin-11 Is an Important Modulator of Retinal Pigment Epithelial Cell Phagocytosis and Cytokine Production.

Science.gov (United States)

Dong, Xia; Wu, Weiju; Ma, Liang; Liu, Chengfei; Bhuckory, Mohajeet B; Wang, Liping; Nandrot, Emeline F; Xu, Heping; Li, Ke; Liu, Yizhi; Zhou, Wuding

2017-01-01

In this paper, we report previously unknown roles for collectin-11 (CL-11, a soluble C-type lectin) in modulating the retinal pigment epithelial (RPE) cell functions of phagocytosis and cytokine production. We found that CL-11 and its carbohydrate ligand are expressed in both the murine and human neural retina; these resemble each other in terms of RPE and photoreceptor cells. Functional analysis of murine RPE cells showed that CL-11 facilitates the opsonophagocytosis of photoreceptor outer segments and apoptotic cells, and also upregulates IL-10 production. Mechanistic analysis revealed that calreticulin on the RPE cells is required for CL-11-mediated opsonophagocytosis whereas signal-regulatory protein α and mannosyl residues on the cells are involved in the CL-11-mediated upregulation of IL-10 production. This study is the first to demonstrate the role of CL-11 and the molecular mechanisms involved in modulating RPE cell phagocytosis and cytokine production. It provides a new insight into retinal health and disease and has implications for other phagocytic cells. © 2017 S. Karger AG, Basel.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

Science.gov (United States)

Okahashi, Nobuo; Okinaga, Toshinori; Sakurai, Atsuo; Terao, Yutaka; Nakata, Masanobu; Nakashima, Keisuke; Shintani, Seikou; Kawabata, Shigetada; Ooshima, Takashi; Nishihara, Tatsuji

2011-10-01

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk

Energy Technology Data Exchange (ETDEWEB)

Yan, Shoubao [Huainan Normal Univ., Anhui (China). School of Life Science; Chen, Xiangsong; Wu, Jingyong; Wang, Pingchao [Chinese Academy of Sciences, Hefei (China). Key Lab. of Ion Beam Bio-engineering of Inst. of Plasma Physics

2012-05-15

The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 {+-} 1.86 g/l, an optimal ethanol concentration of 87.91 {+-} 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h. (orig.)

Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

Science.gov (United States)

Ding, Kai; Han, Lei; Zong, Huifang; Chen, Junsheng; Zhang, Baohong; Zhu, Jianwei

2017-03-01

Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Ca-alginate beads.

Science.gov (United States)

Mohapatra, P K D; Mondal, K C; Pati, B R

2007-06-01

The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation. The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape. The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.

Biotechnological production of pharmaceuticals and biopharmaceuticals in plant cell and organ cultures.

Science.gov (United States)

Hidalgo, Diego; Sanchez, Raul; Lalaleo, Liliana; Bonfill, Mercedes; Corchete, Purificacion; Palazon, Javier

2018-03-09

Plant biofactories are biotechnological platforms based on plant cell and organ cultures used for the production of pharmaceuticals and biopharmaceuticals, although to date only a few of these systems have successfully been implemented at an industrial level. Metabolic engineering is possibly the most straightforward strategy to boost pharmaceutical production in plant biofactories, but social opposition to the use of GMOs means empirical approaches are still being used. Plant secondary metabolism involves thousands of different enzymes, some of which catalyze specific reactions, giving one product from a particular substrate, whereas others can yield multiple products from the same substrate. This trait opens plant cell biofactories to new applications, in which the natural metabolic machinery of plants can be harnessed for the bioconversion of phytochemicals or even the production of new bioactive compounds. Synthetic biological pipelines involving the bioconversion of natural substrates into products with a high market value may be established by the heterologous expression of target metabolic genes in model plants. To summarize the state of the art of plant biofactories and their applications for the pipeline production of cosme-, pharma- and biopharmaceuticals. In order to demonstrate the great potential of plant biofactories for multiple applications in the biotechnological production of pharmaceuticals and biopharmaceuticals, this review broadly covers the following: plant biofactories based on cell and hairy root cultures; secondary metabolite production; biotransformation reactions; metabolic engineering tools applied in plant biofactories; and biopharmaceutical production. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Trichomonas vaginalis α-Actinin 2 Modulates Host Immune Responses by Inducing Tolerogenic Dendritic Cells via IL-10 Production from Regulatory T Cells.

Science.gov (United States)

Lee, Hye-Yeon; Kim, Juri; Ryu, Jae-Sook; Park, Soon-Jung

2017-08-01

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2, Tvα-actinin 2, has been used to diagnose trichomoniasis. This study was undertaken to examine the role of Tvα-actinin 2 as an antigenic molecule to induce immune responses from humans. Western blot analysis using anti-Tvα-actinin 2 antibodies indicated its presence in the secreted proteins of T. vaginalis. ELISA was employed to measure cytokine production by vaginal epithelial cells, prostate cells, mouse dendritic cells (DCs), or T cells stimulated with T. vaginalis or Tvα-actinin 2 protein. Both T. vaginalis and rTvα-actinin 2 induced cytokine production from epithelial cell lines, including IL-10. Moreover, CD4+CD25- regulatory T cells (Treg cells) incubated with rTvα-actinin 2-treated DCs produced high levels of IL-10. These data indicate that Tvα-actinin 2 modulates immune responses via IL-10 production by Treg cells.

The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production.

Science.gov (United States)

Akgul, Yucel; Derk, Raymond C; Meighan, Terence; Rao, K Murali Krishna; Murono, Eisuke P

2011-07-01

The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum. Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17 β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors. Published by Elsevier Inc.

Interleukin-2 production by human leukemia cell lines of pre-B cell origin

International Nuclear Information System (INIS)

Holan, V.; Minowada, J.

1993-01-01

Cells of 7 tested human leukemia cell lines of pre-B cell origin (as characterized by immunophenotyping and by the expression of cytoplasmic micro chains, but not by surface immunoglobulins) produced after stimulation with bacterial lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) a lymphokine activity which supported the growth of the interleukin-2 (IL-2)-dependent CTLL-2 cell line. Three pieces of evidence indicate that the secreted lymphokine was functionally and antigenically very similar, if not identical, to human IL-2: (1) The lymphokine supported the growth of murine IL-2-dependent CTLL-2 cells, which did not respond to human lymphokines other than IL-2, but it did not stimulate the growth of murine IL-3-dependent FDC-P2 cells, (2) the biological activity of the lymphokine was was inhibited by monoclonal antibody (mAb) anti-human-IL-2, and (3) the proliferation of IL-2-dependent cells in the presence of the active materials was completely inhibited by the inclusion of the anti-mouse-IL-2 receptor (IL-2R) mAb. Since leukemia cells of immature B-cell origin also synthesize IL-2R, the human pre-B cell leukemias could represent another type of hematological malignancy where the autocrine processes of IL-2 production and utilization are involved in the expansion of the disease. (author)

Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

Science.gov (United States)

Bodel, P

1978-05-01

Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.

FPGA based Control of a Production Cell System

NARCIS (Netherlands)

Groothuis, M.A.; van Zuijlen, Jasper J.P.; Broenink, Johannes F.

Most motion control systems for mechatronic systems are implemented on digital computers. In this paper we present an FPGA based solution implemented on a low cost Xilinx Spartan III FPGA. A Production Cell setup with multiple parallel operating units is chosen as a test case. The embedded control

Production of L-DOPA by suspension grown cells of Mucuna pruriens.

NARCIS (Netherlands)

Wichers, H.J.

1985-01-01

In vitro cultured plant cells can be utilized for the production of valuable metabolites. Many biochemical and physiological characteristics of ln vitro cultured plant cells depend on various environmental parameters, such as the composition of the growth medium in which many parameters are

Clozapine inhibits Th1 cell differentiation and causes the suppression of IFN-γ production in peripheral blood mononuclear cells.

Science.gov (United States)

Chen, Mao-Liang; Tsai, Tzung-Chieh; Wang, Lu-Kai; Lin, Yi-Yin; Tsai, Ya-Min; Lee, Ming-Cheng; Tsai, Fu-Ming

2012-08-01

Antipsychotic drugs (APDs) are widely used to alleviate a number of psychic disorders and may have immunomodulatory effects. However, the previous studies of cytokine and immune regulation in APDs are quite inconsistent. The aim of this study was to examine the in vitro effects of different ADPs on cytokine production by peripheral blood mononuclear cells (PBMCs). We examined the effects of risperidone, clozapine, and haloperidol on the production of phorbol myristate acetate and ionomycin-induced interferon-γ (IFN-γ)/interleukin (IL)-4 in PBMCs by using intracellular staining. Real-time quantitative PCR and Western blot were used to further examine the expression changes of some critical transcription factors related to T-cell differentiation in antipsychotic-treated PBMCs. Our results indicated that clozapine can suppress the stimulated production of IFN-γ by 30.62%, whereas haloperidol weakly enhances the expression of IFN-γ. Differences in IL-4 production or in the number of CD4+ T cells were not observed in cells treated with different APDs. Furthermore, clozapine and risperidone inhibited the T-bet mRNA and protein expression, which are critical to Th1 differentiation. Also, clozapine can enhance the expression of Signal Transducer and Activator of Transcription 6 and GATA3, which are critical for the differentiation of Th2 cells. The results suggested that clozapine and haloperidol may induce different immunomodulatory effects on the immune system.

Enhancement of Shikonin Production in Suspension Cultures of Lithospermum erythrorhizon Cells by Gamma-irradiation

International Nuclear Information System (INIS)

Baek, Myung Hwa; Chung, Byung Yeoup; Kim, Jae Sung; An, Beyoung Chul; Lee, Young Bok

2005-01-01

The shikonin and several derivatives produced by the roots of Boraginacae family plants are purple compounds that have been used in several parts of the World as antimicrobial and antitumor agents in human pharmaceuticals. Shikonin has been reported as the most successful specimen of the mass production of plant secondary metabolites by cell suspension culture. Numerous studies have elucidated the regulation of production of these compounds in cell suspension cultures. It has known that ultrasonic and gamma irradiation can enhance the production of secondary metabolites. Thus, in present study, we investigate the effects of gamma-irradiation on the shikonin production and the key enzymes in the shikonin biosynthetic pathway of L. erythrorhizon cells

Cell Therapy and Tissue Engineering Products for Chondral Knee Injuries

Directory of Open Access Journals (Sweden)

Adriana Flórez Cabrera

2017-07-01

Full Text Available The articular cartilage is prone to suffer lesions of different etiology, being the articular cartilage lesions of the knee the most common. Although most conventional treatments reduce symptoms they lead to the production of fibrocartilage, which has different characteristics than the hyaline cartilage of the joint. There are few therapeutic approaches that promote the replacement of damaged tissue by functional hyaline cartilage. Among them are the so-called advanced therapies, which use cells and tissue engineering products to promote cartilage regeneration. Most of them are based on scaffolds made of different biomaterials, which seeded or not with endogenous or exogenous cells, can be used as cartilage artificial replacement to improve joint function. This paper reviews some therapeutic approaches focused on the regeneration of articular cartilage of the knee and the biomaterials used to develop scaffolds for cell therapy and tissue engineering of cartilage.

IL-6 Triggers IL-21 production by human CD4(+) T cells to drive STAT3-dependent plasma cell differentiation in B cells

NARCIS (Netherlands)

Diehl, Sean A.; Schmidlin, Heike; Nagasawa, Maho; Blom, Bianca; Spits, Hergen

2012-01-01

Interleukin (IL)-21-producing CD4(+) T cells are central to humoral immunity. Deciphering the signals that induce IL-21 production in CD4(+) T cells and those triggered by IL-21 in B cells are, therefore, of importance for understanding the generation of antibody (Ab) responses. Here, we show that

PAPER PRINTED PHOTOVOLTAIC CELLS: EMERGING METHOD OF PV CELL PRODUCTION

OpenAIRE

Nikhil S. Mane*, Avinash M. Patil2, Vishal P. Patil3

2017-01-01

An Solar energy is a renewable method for the energy production. The use of solar energy is increasing day by day and share of solar energy is increasing in the power sector of India. But as per pollution increases with energy consumption the need of solar energy will goes on increase in recent future as solar energy is a best option in both thermal and photovoltaic energy conversion processes. Photovoltaic cells are compact and has no movable parts which provides them effectiveness and easy ...

Specificity of Good Manufacturing Practice (GMP) for Biomedical Cell Products.

Science.gov (United States)

Tulina, M A; Pyatigorskaya, N V

2018-03-01

The article describes special aspects of Good Manufacturing Practice (GMP) for biomedical cell products (BMCP) that imply high standards of aseptics throughout the entire productio process, strict requirements to donors and to the procedure of biomaterial isolation, guaranty of tracing BMCP products, defining processing procedures which allow to identify BMCP as minimally manipulated; continuous quality control and automation of the control process at all stages of manufacturing, which will ensure product release simultaneously with completion of technological operations.

Inflammation-Induced Changes in Circulating T-Cell Subsets and Cytokine Production During Human Endotoxemia

DEFF Research Database (Denmark)

Ronit, Andreas; Plovsing, Ronni R; Gaardbo, Julie C

2017-01-01

administration. The frequency of anti-inflammatory Tregs increased (P = .033), whereas the frequency of proinflammatory CD4(+)CD161(+) cells decreased (P = .034). Endotoxemia was associated with impaired whole-blood production of tumor necrosis factor-α, interleukin-10, IL-6, IL-17, IL-2, and interferon......Observational clinical studies suggest the initial phase of sepsis may involve impaired cellular immunity. In the present study, we investigated temporal changes in T-cell subsets and T-cell cytokine production during human endotoxemia. Endotoxin (Escherichia coli lipopolysaccharide 4 ng......, HLA-DR(+)CD38(+) T cells were determined. Ex vivo whole-blood cytokine production and Toll-like receptor (TLR)-4 expression on Tregs were measured. Absolute number of CD3(+)CD4(+) (P = .026), CD3(+)CD8(+) (P = .046), Tregs (P = .023), and CD4(+)CD161(+) cells (P = .042) decreased after endotoxin...

The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production

Directory of Open Access Journals (Sweden)

Pornanong Aramwit

2010-05-01

Full Text Available Silk sericin (SS can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells.

Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines.

Science.gov (United States)

Zawada, James F; Yin, Gang; Steiner, Alexander R; Yang, Junhao; Naresh, Alpana; Roy, Sushmita M; Gold, Daniel S; Heinsohn, Henry G; Murray, Christopher J

2011-07-01

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins. Copyright © 2011 Wiley Periodicals, Inc.

Modulation of immunoglobulin production by invariant Vα19-Jα33 TCR-bearing cells.

Directory of Open Access Journals (Sweden)

Michio Shimamura

Full Text Available We have previously shown that invariant Vα19-Jα33 TCR(+ (Vα19i T cells suppress the disease progress in some models for organ specific autoimmune diseases and type IV allergy that deteriorate along with decline to excess in Th1- or Th17- immunity. In this study, we examined the effects of over-generation of Vα19i T cells on the Th2-controlled immunoglobulin isotype production in the models for type I allergy. IgE production by invariant Vα19-Jα33 TCR transgenic (Tg mice was suppressed compared with that by non-Tg controls following administration with goat anti-mouse IgD antiserum or OVA, while IgG2a production was not influenced by the introduction of the transgene into the recipients. IgE production by wild type mice was similarly reduced when they were subjected to adoptive transfer with invariant Vα19-Jα33 TCR Tg(+ but not Tg(- cells prior to immunization. Furthermore, the suppression of IgE production by these recipients was enhanced when they were previously administered with a Vα19i T cell activator, one of the modified α-mannosyl ceramides. In summary, it is suggested that Vα19i T cells have potential to participate in the homeostasis of immunity and that they suppress disease progression resulting from not only Th1- but also Th2- immunity excess.

Flavonoid Production, Growth and Differentiation of Stelechocarpus burahol (Bl.) Hook. F. and Th. Cell Suspension Culture.

Science.gov (United States)

Aini Habibah, Noor; Moeljopawiro, Sukarti; Dewi, Kumala; Indrianto, Ari

2017-01-01

Stelechocarpus burahol is a plant containing flavonoid compounds that have the potential for use as an antihyperuricemic for gout medication. This study was performed to assess flavonoid production, growth and cell differentiation of S. burahol in cell suspension culture. Mesocarp was planted in Murashige and Skoog (MS) medium supplemented with 7.5 mg L-1 picloram for the induction of callus. Non-embryonic callus obtained was used in the formation of cell suspension cultures. Growth of cells was determined by fresh and dry weights. During the culturing, the fresh weight, dry weight and flavonoid content were determined as a result of culture status. The growth of the S. burahol cell suspension was slow, the stationary phase occurred at 30 days. The production of flavonoids was not in line with the growth of cells and the maximum production occurred on the 15th day of the log phase. The globular-shaped cells dominated the cell suspension culture at all ages. Fluorescein diacetate (FDA) staining of cells derived from cell cultures aged for 36 days showed that some cells were still viable. The results show that flavonoid production, growth and cell differentiation of a S. burahol cell suspension culture differed according to the culture age.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Science.gov (United States)

Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

2016-01-01

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Directory of Open Access Journals (Sweden)

Sandeep N Shah

Full Text Available Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

A comparative evaluation of different types of microbial electrolysis desalination cells for malic acid production.

Science.gov (United States)

Liu, Guangli; Zhou, Ying; Luo, Haiping; Cheng, Xing; Zhang, Renduo; Teng, Wenkai

2015-12-01

The aim of this study was to investigate different microbial electrolysis desalination cells for malic acid production. The systems included microbial electrolysis desalination and chemical-production cell (MEDCC), microbial electrolysis desalination cell (MEDC) with bipolar membrane and anion exchange membrane (BP-A MEDC), MEDC with bipolar membrane and cation exchange membrane (BP-C MEDC), and modified microbial desalination cell (M-MDC). The microbial electrolysis desalination cells performed differently in terms of malic acid production and energy consumption. The MEDCC performed best with the highest malic acid production rate (18.4 ± 0.6 mmol/Lh) and the lowest energy consumption (0.35 ± 0.14 kWh/kg). The best performance of MEDCC was attributable to the neutral pH condition in the anode chamber, the lowest internal resistance, and the highest Geobacter percentage of the anode biofilm population among all the reactors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis.

Science.gov (United States)

Kwon, Eun-Yeong; Kim, Kyung Mi; Kim, Mi Kyoung; Lee, In Young; Kim, Beom Soo

2011-09-01

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.

Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

Science.gov (United States)

Zhou, Xiangjun; Zhang, Wei; Yao, Qisheng; Zhang, Hao; Dong, Guie; Zhang, Ming; Liu, Yutao; Chen, Jian-Kang; Dong, Zheng

2017-06-01

Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing. Copyright © 2017 the American Physiological Society.

Production Methods for a Mesenchymal Stem Cell Therapeutic as a Medical Defense Countermeasure

Science.gov (United States)

2012-02-01

mesenchymal stem cell (MSC) efficacy in a variety of injury models demonstrate the unique qualities of this reparative cell population to adapt to the...therapeutic product. Characterization of stem cell properties of culture-expanded MSCs is shown by in vitro differentiation to form mature cell types. The

Ethanol production from molasses by immobilized cells of zymomonas mobilis EMCC 1546

International Nuclear Information System (INIS)

Meliegy, S.A.; Abdelaziz, A.H.

2004-01-01

Ethanol production from beet molasses by zymomonas mobilis EMCC 1546 was studied using continuous processes in which immobilized bacterial cells of Z.mobilis EMCC 1546 was grown on both sodium alginate and polyvinyl alcohol(PVA). The fermentation was performed in a shaking incubation and 1-liter ferment or with final working 750 ml. The initial sugar concentration studied was 50, 100,150, 200 and 250 g/l. The results showed that optimum initial sugar for ethanol production was 200 g/l. In batch fermentation, the highest ethanol concentration was 28.50 g/. Also effect of gamma irradiation was studied to enhance ethanol production. The highest ethanol production at dose dose 0.25 kGy was 34.82 g/l. The results showed that continuous fermentation, at dilution rate 1.36 (I/h), helped to increase the ethanol production significantly and continuous fermentation with immobilized cells in alginate gave higher ethanol production, 35.8 (g/I), as compared with those immobilized in hydrogel (PVA)

Viability of fuel cells for car production

Energy Technology Data Exchange (ETDEWEB)

Buchel, J.-P. [Renault, Trappes (France); Lisse, J.-P. [P.S.A., Trappes (France); Bernard, S. [Alten, Trappes (France)

2000-07-01

The two French car manufacturers PSA Peugeot Citroen and Renault both sell pure electric cars in an effort to reduce pollutants and carbon dioxide emissions. In addition, they have each studied fuel cell car prototypes in relation to the FEVER program for Renault and the HYDRO-GEN program for PSA. In 1999, the two manufacturers joined forces in a common program to evaluate the technical, economical and environmental viability of the fuel cell vehicle potential. The joint program has active contributions by Air Liquid, the French Atomic Energy Agency, De Nora Fuel Cells, Elf-Antar-France, Totalfina and Valeo. This paper highlighted many of the components of this program and the suitability of this new technology for industrial production at a cost competitive price. Certain automotive constraints have to be considered to propose vehicles which could provide good performance in varying temperature and operating conditions. Safety is also an important concern given that the vehicles are powered by hydrogen and a high voltage power source. Another challenges is the choice of the fuel and the economic cost of a new refueling infrastructure. Recycling was suggested as a means to recover expensive fuel cell system components such as precious catalysts, bipolar plates, membranes and other main specific parts of the fuel cell vehicle. This paper also discussed issues regarding the thermal management of the fuel cell power plant and air conditioning of the vehicles. figs.

Protective role of melatonin in progesterone production by human luteal cells.

Science.gov (United States)

Taketani, Toshiaki; Tamura, Hiroshi; Takasaki, Akihisa; Lee, Lifa; Kizuka, Fumie; Tamura, Isao; Taniguchi, Ken; Maekawa, Ryo; Asada, Hiromi; Shimamura, Katsunori; Reiter, Russel J; Sugino, Norihiro

2011-09-01

This study investigated whether melatonin protects luteinized granulosa cells from reactive oxygen species (ROS) as an antioxidant to enhance progesterone production in the follicle during ovulation. Follicular fluid was sampled at the time of oocyte retrieval in women undergoing in vitro fertilization and embryo transfer (IVF-ET). Melatonin concentrations in the follicular fluid were positively correlated with progesterone concentrations (r = 0.342, P progesterone and 8-OHdG concentrations were negatively correlated (r = -0.246, P Progesterone production by luteinized granulosa cells was significantly inhibited by H(2)O(2). Melatonin treatment overcame the inhibitory effect of H(2) O(2) . Twenty-five patients who had luteal phase defect (serum progesterone concentrations progesterone concentrations (>10 ng/mL during the mid-luteal phase) in nine of 14 women (64.3%), whereas only two of 11 women (18.1%) showed normal serum progesterone levels in the control group. In conclusion, melatonin protects granulosa cells undergoing luteinization from ROS in the follicle and contributes to luteinization for progesterone production during ovulation. © 2011 John Wiley & Sons A/S.

Cost Analysis of Direct Methanol Fuel Cell Stacks for Mass Production

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Mauro Francesco Sgroi

2016-11-01

Full Text Available Fuel cells are very promising technologies for efficient electrical energy generation. The development of enhanced system components and new engineering solutions is fundamental for the large-scale deployment of these devices. Besides automotive and stationary applications, fuel cells can be widely used as auxiliary power units (APUs. The concept of a direct methanol fuel cell (DMFC is based on the direct feed of a methanol solution to the fuel cell anode, thus simplifying safety, delivery, and fuel distribution issues typical of conventional hydrogen-fed polymer electrolyte fuel cells (PEMFCs. In order to evaluate the feasibility of concrete application of DMFC devices, a cost analysis study was carried out in the present work. A 200 W-prototype developed in the framework of a European Project (DURAMET was selected as the model system. The DMFC stack had a modular structure allowing for a detailed evaluation of cost characteristics related to the specific components. A scale-down approach, focusing on the model device and projected to a mass production, was used. The data used in this analysis were obtained both from research laboratories and industry suppliers specialising in the manufacturing/production of specific stack components. This study demonstrates that mass production can give a concrete perspective for the large-scale diffusion of DMFCs as APUs. The results show that the cost derived for the DMFC stack is relatively close to that of competing technologies and that the introduction of innovative approaches can result in further cost savings.

Astaxanthin increases progesterone production in cultured bovine luteal cells.

Science.gov (United States)

Kamada, Hachiro; Akagi, Satoshi; Watanabe, Shinya

2017-06-29

Although astaxanthin (AST) is known to be a strong antioxidant, its effects on reproductive function in domestic animals have not yet been elucidated in detail. Therefore, we investigated the effects of AST on luteal cells, which produce progesterone (P4), an important hormone for maintaining pregnancy. Luteal cells were prepared by collagenase dispersion of the corpus luteum (CL). The addition of racemic AST at a low concentration (production than RR-AST. When 1 mg/kg·body weight of SS-AST derived from green algae was fed to cows for 2 weeks, its concentration in blood plasma was 10.9 nM on average, which was sufficient to expect an in vitro effect on the production of P4 in cows. These results suggested the potential of SS-AST supplements for cows to elevate luteal function.

Hydrogen production through high-temperature electrolysis in a solid oxide cell

International Nuclear Information System (INIS)

Herring, J.St.; Lessing, P.; O'Brien, J.E.; Stoots, C.; Hartvigsen, J.; Elangovan, S.

2004-01-01

An experimental research programme is being conducted by the INEEL and Ceramatec, Inc., to test the high-temperature, electrolytic production of hydrogen from steam using a solid oxide cell. The research team is designing and testing solid oxide cells for operation in the electrolysis mode, producing hydrogen rising a high-temperature heat and electrical energy. The high-temperature heat and the electrical power would be supplied simultaneously by a high-temperature nuclear reactor. Operation at high temperature reduces the electrical energy requirement for electrolysis and also increases the thermal efficiency of the power-generating cycle. The high-temperature electrolysis process will utilize heat from a specialized secondary loop carrying a steam/hydrogen mixture. It is expected that, through the combination of a high-temperature reactor and high-temperature electrolysis, the process will achieve an overall thermal conversion efficiency of 40 to 50%o while avoiding the challenging chemistry and corrosion issues associated with the thermochemical processes. Planar solid oxide cell technology is being utilised because it has the best potential for high efficiency due to minimized voltage and current losses. These losses also decrease with increasing temperature. Initial testing has determined the performance of single 'button' cells. Subsequent testing will investigate the performance of multiple-cell stacks operating in the electrolysis mode. Testing is being performed both at Ceramatec and at INEEL. The first cells to be tested were single cells based on existing materials and fabrication technology developed at Ceramatec for production of solid oxide fuel cells. These cells use a relatively thick (∼ 175 μm) electrolyte of yttria- or scandia-stabilised zirconia, with nickel-zirconia cermet anodes and strontium-doped lanthanum manganite cathodes. Additional custom cells with lanthanum gallate electrolyte have been developed and tested. Results to date have

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

Science.gov (United States)

Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

2018-03-03

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

Continuous ethanol production from Jerusalem artichokes stalks using immobilized cells of Kluyveromyces marxianus

Energy Technology Data Exchange (ETDEWEB)

Bajpai, P.; Margaritis, A.

1986-01-01

Continuous production of ethanol from the extract of Jerusalem artichoke stalks was investigated in a packed bed bioreactor using Kluyveromyces marxianus cells immobilized in calcium alginate gel beds. Maximum conversion of the sugars to ethanol was achieved with a yield of about 98% of the theoretical. Volumetric ethanol productivities of 102 grams of ethanol per litre per hour and 92 grams ethanol per liter per hour were obtained at 87% and 90% conversion respectively for an inlet substrate concentration of 100 gram sugars per liter. The maximum specific ethanol production rate and maximum specific total sugar uptake rate of the immobilized cells were found to be 0.96 gram ethanol per gram immobilized cells per hour and 2.06 gram sugars per gram immobilized cells per hour respectively. The immobilized cell bioreactor was run continuously at a dilution rate of 2.12 per hour for 30 days which resulted in a loss of 30% of the original activity. The half life of the bioreactor was estimated to be about 56 days.

Yeast cell metabolism investigated by CO{_2} production and soft X-ray irradiation

Science.gov (United States)

Masini, A.; Batani, D.; Previdi, F.; Milani, M.; Pozzi, A.; Turcu, E.; Huntington, S.; Takeyasu, H.

1999-01-01

Results obtained using a new technique for studying cell metabolism are presented. The technique, consisting in CO2 production monitoring, has been applied to Saccharomyces cerevisiae yeast cells. Also the cells were irradiated using the soft X-ray laser-plasma source at Rutherford Appleton Laboratory with the aim of producing a damage of metabolic processes at the wall level, responsible for fermentation, without great interference with respiration, taking place in mitochondria, and DNA activity. The source was calibrated with PIN diodes and X-ray spectrometers and used Teflon stripes as target, emitting X-rays at about 0.9 keV, with a very low penetration in biological material. X-ray doses delivered to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. Immediately after irradiation, the damage to metabolic activity was measured again by monitoring CO2 production. Results showed a general reduction in gas production by irradiated samples, together with non-linear and non-monotone response to dose. There was also evidence of oscillations in cell metabolic activity and of X-ray induced changes in oscillation frequency.

The response of CD1d-restricted invariant NKT cells to microbial pathogens and their products

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Luc eVan Kaer

2015-05-01

Full Text Available Invariant natural killer T (iNKT cells become activated during a wide variety of infections. This includes organisms lacking cognate CD1d-binding glycolipid antigens recognized by the semi-invariant T cell receptor of iNKT cells. Additional studies have shown that iNKT cells also become activated in vivo in response to microbial products such as bacterial lipopolysaccharide, a potent inducer of cytokine production in antigen-presenting cells (APCs. Other studies have shown that iNKT cells are highly responsive to stimulation by cytokines such as interleukin-12. These findings have led to the concept that microbial pathogens can activate iNKT cells either directly via glycolipids, or indirectly by inducing cytokine production in APCs. iNKT cells activated in this manner produce multiple cytokines that can influence the outcome of infection, usually in favor of the host, although potent iNKT cell activation may contribute to an uncontrolled cytokine storm and sepsis. One aspect of the response of iNKT cells to microbial pathogens is that it is short-lived and followed by an extended time period of unresponsiveness to reactivation. This refractory period may represent a means to avoid chronic activation and cytokine production by iNKT cells, thus protecting the host against some of the negative effects of iNKT cell activation, but potentially putting the host at risk for secondary infections. These effects of microbial pathogens and their products on iNKT cells are not only important for understanding the role of these cells in immune responses against infections but also for the development of iNKT cell-based therapies.

Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

African Journals Online (AJOL)

Enhancement of Diosgenin Production in Plantlet and Cell Cultures of Dioscorea zingiberensis by Palmarumycin C13 from the Endophytic fungus, Berkleasmium sp. Dzf12. Y Mou, K Zhou, D Xu, R Yu, J Li, C Yin, L Zhou ...

Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

Science.gov (United States)

Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

2014-02-01

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

Enzyme production in immobilized Trichoderma reesei cells with hydrophobic polymers prepared by radiation polymerization method

International Nuclear Information System (INIS)

Luzhao Xin; Kumakura, Minoru; Kaetsu, Isao

1993-01-01

Trichoderma reesei cells were immobilized on paper covered with hydrophobic monomer, trimethylpropane triacrylate by radiation polymerization. The effect of immobilization condition on enzyme productivity was studied by measuring filter paper and cellobiose activity. The cells were adhered and grew on the surface of the carrier with the polymer giving high enzyme productivity in the immobilized cells in comparison with the free cells. Optimum concentration and volume of the coating monomer for the preparation of the immobilized cells were obtained. (author)

β-cryptoxanthin regulates bone resorption related-cytokine production in human periodontal ligament cells.

Science.gov (United States)

Nishigaki, Masaru; Yamamoto, Toshiro; Ichioka, Hiroaki; Honjo, Ken-Ichi; Yamamoto, Kenta; Oseko, Fumishige; Kita, Masakazu; Mazda, Osam; Kanamura, Narisato

2013-07-01

β-cryptoxanthin (β-cry) is a type of carotenoid found in certain fruits and vegetables. Although it has been shown that β-cry inhibits alveolar bone resorption, the molecular mechanisms for this have not yet been clarified. In the present study, we investigated the effects of β-cry on bone resorption related-cytokine production in human periodontal ligament (hPDL) cells. hPDL cells were stimulated with β-cry (1×10(-7)mol/l), mechanical stress (1 or 6MPa), and P. gingivalis. The production of interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by RT-PCR and ELISA. The production of IL-1β, IL-6, IL-8, and TNF-α was not induced in hPDL cells after stimulation with β-cry, although these cytokines were produced after stimulation with P. gingivalis. On the other hand, IL-6 and IL-8 were produced after exposure to 6MPa of mechanical stress. The production of IL-6 and IL-8 was significantly decreased by the addition of β-cry. Furthermore, β-cry up-regulated the production of OPG, but not RANKL. β-cry inhibited the production of IL-6 and IL-8 induced by mechanical stress and periodontopathogenic bacteria in hPDL cells. Moreover, β-cry up-regulated OPG production. These results suggest that β-cry may prevent bone resorption in periodontitis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals.

Science.gov (United States)

Butler, Michael

2005-08-01

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number

Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Mycobacterium tuberculosis Infection.

Science.gov (United States)

Rothchild, Alissa C; Stowell, Britni; Goyal, Girija; Nunes-Alves, Cláudio; Yang, Qianting; Papavinasasundaram, Kadamba; Sassetti, Christopher M; Dranoff, Glenn; Chen, Xinchun; Lee, Jinhee; Behar, Samuel M

2017-10-24

Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF -/- ) are highly susceptible to infection with Mycobacterium tuberculosis , and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4 + T cells as the infection progresses. M. tuberculosis -specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis IMPORTANCE Mycobacterium tuberculosis is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune

Effect of Lipopolysaccharide on Progesterone Production during Luteinization of Granulosa and Theca cells In Vitro.

Science.gov (United States)

Shimizu, Takashi; Echizenya, Riku; Miyamoto, Akio

2016-04-01

The aim of this study is to examine the effect of lipopolysaccharide (LPS) on progesterone production during luteinization of granulosa and theca cells isolated from bovine large follicles. Granulosa and theca cells isolated from large follicles of bovine ovaries were exposed to LPS under appropriate hormone conditions in vitro. Progesterone (P4) production in theca cells, but not granulosa cells, was decreased by long-term exposure of LPS. Long-term exposure of LPS suppressed the gene expression of luteinizing hormone receptor in theca cells. Although long-term exposure of LPS did not affect the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) genes, it did inhibit the protein expression of StAR and 3β-HSD in theca cells. These findings suggest that theca cells, rather than granulosa cells, are susceptible to LPS during luteinization and that LPS inhibits P4 production by decreasing protein levels of StAR during luteinization of theca cells. © 2016 Wiley Periodicals, Inc.

Controlling cell volume for efficient PHB production by Halomonas.

Science.gov (United States)

Jiang, Xiao-Ran; Yao, Zhi-Hao; Chen, Guo-Qiang

2017-11-01

Bacterial morphology is decided by cytoskeleton protein MreB and cell division protein FtsZ encoded by essential genes mreB and ftsZ, respectively. Inactivating mreB and ftsZ lead to increasing cell sizes and cell lengths, respectively, yet seriously reduce cell growth ability. Here we develop a temperature-responsible plasmid expression system for compensated expression of relevant gene(s) in mreB or ftsZ disrupted recombinants H. campaniensis LS21, allowing mreB or ftsZ disrupted recombinants to grow normally at 30°C in a bioreactor for 12h so that a certain cell density can be reached, followed by 36h cell size expansions or cell shape elongations at elevated 37°C at which the mreB and ftsZ encoded plasmid pTKmf failed to replicate in the recombinants and thus lost themselves. Finally, 80% PHB yield increase was achieved via controllable morphology manipulated H. campaniensis LS21. It is concluded that controllable expanding cell volumes (widths or lengths) provides more spaces for accumulating more inclusion body polyhydroxybutyrate (PHB) and the resulting cell gravity precipitation benefits the final separation of cells and product during downstream. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Parameters for MDBK cell growth on microcarriers and BoHV-1 virus production

Directory of Open Access Journals (Sweden)

Ethel C. Freitas

2017-09-01

Full Text Available Bovine herpes virus 1 (BoHV-1 is an important veterinary agent , which causes infectious bovine rhinotra-cheitis. This disease affects the respiratory tract or genitals, causing weight loss, reduced milk production and abor-tion. Several vaccines against BoHV-1 have been developed. In this paper, we study the parameters for MDBK growth on microcarriers (Cytodex 1 and for BoHV-1 virus production. The cell culture attached to microcarriers is an effi-cient method to enlarge the surface of cell growth and for large-scale cell production. Our studies reveal that MDBK adhered to MCs in 30 minutes and that initial agitation of culture did not influence on the efficiency of adhesion or cell growth. In our experiments, we detected no relevant influence of agitation on initial cell adhesion of MDBK to MCs. The maximum cell yield was similar to all initial conditions of agitation studied. The maximum yield obtained in culture started with 15, 20 and 30 cells / MC, was respectively.8.7 x105, 9.3 x105 and 9.8 x105 cells / ml . The cellular distribution on the MCs at the beginning of the culture was more heterogeneous in higher initial densities. After three medium exchanges during MDBK cell culture, the increase in the final yield was 100% higher than that from culture performed without medium change (0.93 x 106 cells / mL. Replacing 50% of the culture medium with fresh medium after 24 hours of growth, the concentration of glucose (5 mM and glutamine (1.8 mM were almost completely res-tored. In these studies, BoHV-1 infections of MDBK were performed after 48, 72 and 86 hours with daily exchanges of 50% of the medium. The increase in viral titer was proportional to the number of viable cells present at the time of infection. The best result of BoHV-1 production was achieved when the infection was performed from 86 hours of cell culture, reaching about 3.7 x108 (TCID50/ml after 24-48 hours of infection, being on average four times higher when compared to the

Advanced process monitoring and feedback control to enhance cell culture process production and robustness.

Science.gov (United States)

Zhang, An; Tsang, Valerie Liu; Moore, Brandon; Shen, Vivian; Huang, Yao-Ming; Kshirsagar, Rashmi; Ryll, Thomas

2015-12-01

It is a common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds, based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply process analytical technology (PAT), a fully automated cell culture feedback control system has been explored in three different applications. The first study illustrates that frequent monitoring and automatically controlling the complex feed based on a surrogate (glutamate) level improved protein production. More importantly, the resulting feed strategy was translated into a manufacturing-friendly manual feed strategy without impact on product quality. The second study demonstrates the improved process robustness of an automated feed strategy based on online bio-capacitance measurements for cell growth. In the third study, glucose and lactate concentrations were measured online and were used to automatically control the glucose feed, which in turn changed lactate metabolism. These studies suggest that the auto-feedback control system has the potential to significantly increase productivity and improve robustness in manufacturing, with the goal of ensuring process performance and product quality consistency. © 2015 Wiley Periodicals, Inc.

Characterizing Enterovirus 71 and Coxsackievirus A16 virus-like particles production in insect cells.

Science.gov (United States)

Somasundaram, Balaji; Chang, Cindy; Fan, Yuan Y; Lim, Pei-Yin; Cardosa, Jane; Lua, Linda

2016-02-15

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two viruses commonly responsible for hand, foot and mouth disease (HFMD) in children. The lack of prophylactic or therapeutic measures against HFMD is a major public health concern. Insect cell-based EV71 and CVA16 virus-like particles (VLPs) are promising vaccine candidates against HFMD and are currently under development. In this paper, the influence of insect cell line, incubation temperature, and serial passaging effect and stability of budded virus (BV) stocks on EV71 and CVA16 VLP production was investigated. Enhanced EV71 and CVA16 VLP production was observed in Sf9 cells compared to High Five™ cells. Lowering the incubation temperature from the standard 27°C to 21°C increased the production of both VLPs in Sf9 cells. Serial passaging of CVA16 BV stocks in cell culture had a detrimental effect on the productivity of the structural proteins and the effect was observed with only 5 passages of BV stocks. A 2.7× higher production yield was achieved with EV71 compared to CVA16. High-resolution asymmetric flow field-flow fractionation couple with multi-angle light scattering (AF4-MALS) was used for the first time to characterize EV71 and CVA16 VLPs, displaying an average root mean square radius of 15±1nm and 15.3±5.8 nm respectively. This study highlights the need for different approaches in the design of production process to develop a bivalent EV71 and CVA16 vaccine. Copyright © 2015 Elsevier Inc. All rights reserved.

NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells

Energy Technology Data Exchange (ETDEWEB)

Shoji, Wataru [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Suenaga, Yusuke, E-mail: ysuenaga@chiba-cc.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Yokoi, Sana [Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Nio, Masaki [Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Nakagawara, Akira, E-mail: nakagawara-a@koseikan.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan)

2015-06-05

NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. - Highlights: • NCYM promotes cleavages of MYC and MYCN to produce Myc-nick in vitro. • NCYM increases Myc-nick production in MYCN-amplified neuroblastoma cells. • NCYM knockdown decreases Myc-nick production and induces apoptosis at G2/M phase.

NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells

International Nuclear Information System (INIS)

Shoji, Wataru; Suenaga, Yusuke; Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer; Yokoi, Sana; Nio, Masaki; Nakagawara, Akira

2015-01-01

NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. - Highlights: • NCYM promotes cleavages of MYC and MYCN to produce Myc-nick in vitro. • NCYM increases Myc-nick production in MYCN-amplified neuroblastoma cells. • NCYM knockdown decreases Myc-nick production and induces apoptosis at G2/M phase

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells.

Science.gov (United States)

Carvalho, Larissa A C; Lopes, João P P B; Kaihami, Gilberto H; Silva, Railmara P; Bruni-Cardoso, Alexandre; Baldini, Regina L; Meotti, Flavia C

2018-06-01

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl - /H 2 O 2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Photocatalysis for Renewable Energy Production Using PhotoFuelCells

Directory of Open Access Journals (Sweden)

Robert Michal

2014-11-01

Full Text Available The present work is a short review of our recent studies on PhotoFuelCells, that is, photoelectrochemical cells which consume a fuel to produce electricity or hydrogen, and presents some unpublished data concerning both electricity and hydrogen production. PhotoFuelCells have been constructed using nanoparticulate titania photoanodes and various cathode electrodes bearing a few different types of electrocatalyst. In the case where the cell functioned with an aerated cathode, the cathode electrode was made of carbon cloth carrying a carbon paste made of carbon black and dispersed Pt nanoparticles. When the cell was operated in the absence of oxygen, the electrocatalyst was deposited on an FTO slide using a special commercial carbon paste, which was again enriched with Pt nanoparticles. Mixing of Pt with carbon paste decreased the quantity of Pt necessary to act as electrocatalyst. PhotoFuelCells can produce electricity without bias and with relatively high open-circuit voltage when they function in the presence of fuel and with an aerated cathode. In that case, titania can be sensitized in the visible region by CdS quantum dots. In the present work, CdS was deposited by the SILAR method. Other metal chalcogenides are not functional as sensitizers because the combined photoanode in their presence does not have enough oxidative power to oxidize the fuel. Concerning hydrogen production, it was found that it is difficult to produce hydrogen in an alkaline environment even under bias, however, this is still possible if losses are minimized. One way to limit losses is to short-circuit anode and cathode electrode and put them close together. This is achieved in the “photoelectrocatalytic leaf”, which was presently demonstrated capable of producing hydrogen even in a strongly alkaline environment.

Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

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Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

2015-12-01

Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

Repletion of zinc in zinc-deficient cells strongly up-regulates IL-1β-induced IL-2 production in T-cells.

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Daaboul, Doha; Rosenkranz, Eva; Uciechowski, Peter; Rink, Lothar

2012-10-01

Mild zinc deficiency in humans negatively affects IL-2 production resulting in declined percentages of cytolytic T cells and decreased NK cell lytic activity, which enhances the susceptibility to infections and malignancies. T-cell activation is critically regulated by zinc and the normal physiological zinc level in T-cells slightly lies below the optimal concentration for T-cell functions. A further reduction in zinc level leads to T-cell dysfunction and autoreactivity, whereas high zinc concentrations (100 μM) were shown to inhibit interleukin-1 (IL-1)-induced IL-1 receptor kinase (IRAK) activation. In this study, we investigated the molecular mechanism by which zinc regulates the IL-1β-induced IL-2 expression in T-cells. Zinc supplementation to zinc-deficient T-cells increased intracellular zinc levels by altering the expression of zinc transporters, particularly Zip10 and Zip12. A zinc signal was observed in the murine T-cell line EL-4 6.1 after 1 h of stimulation with IL-1β, measured by specific zinc sensors FluoZin-3 and ZinPyr-1. This signal is required for the phosphorylation of MAPK p38 and NF-κB subunit p65, which triggers the transcription of IL-2 and strongly increases its production. These results indicate that short-term zinc supplementation to zinc-deficient T-cells leads to a fast rise in zinc levels which subsequently enhance cytokine production. In conclusion, low and excessive zinc levels might be equally problematic for zinc-deficient subjects, and stabilized zinc levels seem to be essential to avoid negative concentration-dependent zinc effects on T-cell activation.

Production of membrane proteins without cells or detergents.

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Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

2011-04-30

The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

Effect of microwell chip structure on cell microsphere production of various animal cells.

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Sakai, Yusuke; Yoshida, Shirou; Yoshiura, Yukiko; Mori, Rhuhei; Tamura, Tomoko; Yahiro, Kanji; Mori, Hideki; Kanemura, Yonehiro; Yamasaki, Mami; Nakazawa, Kohji

2010-08-01

The formation of three-dimensional cell microspheres such as spheroids, embryoid bodies, and neurospheres has attracted attention as a useful culture technique. In this study, we investigated a technique for effective cell microsphere production by using specially prepared microchip. The basic chip design was a multimicrowell structure in triangular arrangement within a 100-mm(2) region in the center of a polymethylmethacrylate (PMMA) plate (24x24 mm(2)), the surface of which was modified with polyethylene glycol (PEG) to render it nonadhesive to cells. We also designed six similar chips with microwell diameters of 200, 300, 400, 600, 800, and 1000 microm to investigate the effect of the microwell diameter on the cell microsphere diameter. Rat hepatocytes, HepG2 cells, mouse embryonic stem (ES) cells, and mouse neural progenitor/stem (NPS) cells formed hepatocyte spheroids, HepG2 spheroids, embryoid bodies, and neurospheres, respectively, in the microwells within 5 days of culture. For all the cells, a single microsphere was formed in each microwell under all the chip conditions, and such microsphere configurations remained throughout the culture period. Furthermore, the microsphere diameters of each type of cell were strongly positively correlated with the microwell diameters of the chips, suggesting that microsphere diameter can be factitiously controlled by using different chip conditions. Thus, this chip technique is a promising cellular platform for tissue engineering or regenerative medicine research, pharmacological and toxicological studies, and fundamental studies in cell biology. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

Science.gov (United States)

Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

2014-01-01

ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4.

Science.gov (United States)

Todaro, Matilde; Alea, Mileidys Perez; Di Stefano, Anna B; Cammareri, Patrizia; Vermeulen, Louis; Iovino, Flora; Tripodo, Claudio; Russo, Antonio; Gulotta, Gaspare; Medema, Jan Paul; Stassi, Giorgio

2007-10-11

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor. Here, we describe the identification and characterization of such cells from colon carcinomas using the stem cell marker CD133 that accounts around 2% of the cells in human colon cancer. The CD133(+) cells grow in vitro as undifferentiated tumor spheroids, and they are both necessary and sufficient to initiate tumor growth in immunodeficient mice. Xenografts resemble the original human tumor maintaining the rare subpopulation of tumorigenic CD133(+) cells. Further analysis revealed that the CD133(+) cells produce and utilize IL-4 to protect themselves from apoptosis. Consistently, treatment with IL-4Ralpha antagonist or anti-IL-4 neutralizing antibody strongly enhances the antitumor efficacy of standard chemotherapeutic drugs through selective sensitization of CD133(+) cells. Our data suggest that colon tumor growth is dictated by stem-like cells that are treatment resistant due to the autocrine production of IL-4.

Solid oxide fuel cells and hydrogen production

International Nuclear Information System (INIS)

Dogan, F.

2009-01-01

'Full text': A single-chamber solid oxide fuel cell (SC-SOFC), operating in a mixture of fuel and oxidant gases, provides several advantages over the conventional SOFC such as simplified cell structure (no sealing required). SC-SOFC allows using a variety of fuels without carbon deposition by selecting appropriate electrode materials and cell operating conditions. The operating conditions of single chamber SOFC was studied using hydrocarbon-air gas mixtures for a cell composed of NiO-YSZ / YSZ / LSCF-Ag. The cell performance and catalytic activity of the anode was measured at various gas flow rates. The results showed that the open-circuit voltage and the power density increased as the gas flow rate increased. Relatively high power densities up to 660 mW/cm 2 were obtained in a SC-SOFC using porous YSZ electrolytes instead of dense electrolytes required for operation of a double chamber SOFC. In addition to propane- or methane-air mixtures as a fuel source, the cells were also tested in a double chamber configuration using hydrogen-air mixtures by controlling the hydrogen/air ratio at the cathode and the anode. Simulation of single chamber conditions in double chamber configurations allows distinguishing and better understanding of the electrode reactions in the presence of mixed gases. Recent research efforts; the effect of hydrogen-air mixtures as a fuel source on the performance of anode and cathode materials in single-chamber and double-chamber SOFC configurations,will be presented. The presentation will address a review on hydrogen production by utilizing of reversible SOFC systems. (author)

Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations.

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Sprenger, Georg A; Baumgärtner, Florian; Albermann, Christoph

2017-09-20

Human milk oligosaccharides (HMO) are almost unique constituents of breast milk and are not found in appreciable amounts in cow milk. Due to several positive aspects of HMO for the development, health, and wellbeing of infants, production of HMO would be desirable. As a result, scientists from different disciplines have developed methods for the preparation of single HMO compounds. Here, we review approaches to HMO preparation by (chemo-)enzymatic syntheses or by whole-cell biotransformation with recombinant bacterial cells. With lactose as acceptor (in vitro or in vivo), fucosyltransferases can be used for the production of 2'-fucosyllactose, 3-fucosyllactose, or more complex fucosylated core structures. Sialylated HMO can be produced by sialyltransferases and trans-sialidases. Core structures as lacto-N-tetraose can be obtained by glycosyltransferases from chemical donor compounds or by multi-enzyme cascades; recent publications also show production of lacto-N-tetraose by recombinant Escherichia coli bacteria and approaches to obtain fucosylated core structures. In view of an industrial production of HMOs, the whole cell biotransformation is at this stage the most promising option to provide human milk oligosaccharides as food additive. Copyright © 2017 Elsevier B.V. All rights reserved.

Autocrine production of beta-chemokines protects CMV-Specific CD4 T cells from HIV infection.

Directory of Open Access Journals (Sweden)

Joseph P Casazza

2009-10-01

Full Text Available Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1alpha and MIP-1beta mRNA, resulting in a rapid increase in production of MIP-1alpha and MIP-1beta after cognate antigen stimulation. Production of beta-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of beta-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1beta contained 10 times less Gag DNA than did those which failed to produce MIP-1beta. These data suggest that CD4+ T cells which produce MIP-1alpha and MIP-1beta bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.

Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

Energy Technology Data Exchange (ETDEWEB)

Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.

2017-05-23

Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

Development and scale-up of the production process of NovoCell fuel cells; Desenvolvimento e 'scale-up' do processo de producao de celulas a combustivel NovoCell

Energy Technology Data Exchange (ETDEWEB)

Azevedo, Dayse Caldas de; Souza, Adler de; Ferreira, Valdemar Stelita [NovoCell Sistemas de Energia S.A., Santa Barbara D' Oeste, SP (Brazil)

2008-07-01

Fuel cells present the potentiality to substitute the engines of internal combustion in vehicles and to supply energy for stationary use. This potentiality, however, not yet reflected in its introduction in the market with regular lines of production, because of its high cost and lack of criteria that demonstrate its reliability and durability. These subjects are the main goals of the programs of development of fuel cells worldwide. NovoCell is a Brazilian company whose objective is to develop and to produce hydrogen/air fuel cells for stationary generation. All the project is guided by the use of technologies/processes and materials that allow production in large scale and to a competitive cost, giving support to a continuous program of innovation and development of the product. In this work the technological solutions developed by the company are presented. (author)

Production Cells in Construction: Considering Time, Space and Information Linkages to Seek Broader Implementations

Directory of Open Access Journals (Sweden)

Renato Nunes Mariz

2013-01-01

Full Text Available The use of production cells in manufacturing has achieved many benefits, motivating researchers to apply them in the construction environment. The aim of this research is to identify time, space, and information linkages in construction’s production cells applications, seeking opportunities for broader implementations. We adopted a literature review approach focusing on cases in the Brazilian construction sector that addressed cell applications. Subsequently, comparative tables of these publications were prepared, analyzing the consideration of time, space, and information linkages, as well as identified results. The article pointed out that there is a gap in publications that address the application of a production cell in almost all construction flows, except the job site flow, reflecting the tendency of most companies of applying lean concepts firstly in physical flows. By analyzing these aspects (group of features that enhance the use of the cell, it was found that “material flow and pull systems” and “operators interaction” were the aspects most often considered, but mostly partially. Few cases reported the use of “flexibility” and “equipment maintenance”. No case reported comprehensive considerations of the three important linkages of time, space, and information. Space was the linkage better considered compared to time and information linkages. Lead time reduction, cost savings and increased productivity were among the greatest benefits reported from the applications of production cells. There is also a positive correlation between the linkages coverage and the number of benefits obtained. Further research is suggested in order to investigate the results of a more comprehensive application considering all linkages.

Final Technical Report Microwave Assisted Electrolyte Cell for Primary Aluminum Production

Energy Technology Data Exchange (ETDEWEB)

Xiaodi Huang; J.Y. Hwang

2007-04-18

This research addresses the high priority research need for developing inert anode and wetted cathode technology, as defined in the Aluminum Industry Technology Roadmap and Inert Anode Roadmap, with the performance targets: a) significantly reducing the energy intensity of aluminum production, b) ultimately eliminating anode-related CO2 emissions, and c) reducing aluminum production costs. This research intended to develop a new electrometallurgical extraction technology by introducing microwave irradiation into the current electrolytic cells for primary aluminum production. This technology aimed at accelerating the alumina electrolysis reduction rate and lowering the aluminum production temperature, coupled with the uses of nickel based superalloy inert anode, nickel based superalloy wetted cathode, and modified salt electrolyte. Michigan Technological University, collaborating with Cober Electronic and Century Aluminum, conducted bench-scale research for evaluation of this technology. This research included three sub-topics: a) fluoride microwave absorption; b) microwave assisted electrolytic cell design and fabrication; and c) aluminum electrowinning tests using the microwave assisted electrolytic cell. This research concludes that the typically used fluoride compound for aluminum electrowinning is not a good microwave absorbing material at room temperature. However, it becomes an excellent microwave absorbing material above 550°C. The electrowinning tests did not show benefit to introduce microwave irradiation into the electrolytic cell. The experiments revealed that the nickel-based superalloy is not suitable for use as a cathode material; although it wets with molten aluminum, it causes severe reaction with molten aluminum. In the anode experiments, the chosen superalloy did not meet corrosion resistance requirements. A nicked based alloy without iron content could be further investigated.

Spatial separation of photosynthesis and ethanol production by cell type-specific metabolic engineering of filamentous cyanobacteria.

Science.gov (United States)

Ehira, Shigeki; Takeuchi, Takuto; Higo, Akiyoshi

2018-02-01

Cyanobacteria, which perform oxygenic photosynthesis, have drawn attention as hosts for the direct production of biofuels and commodity chemicals from CO 2 and H 2 O using light energy. Although cyanobacteria capable of producing diverse chemicals have been generated by metabolic engineering, anaerobic non-photosynthetic culture conditions are often necessary for their production. In this study, we conducted cell type-specific metabolic engineering of the filamentous cyanobacterium Anabaena sp. PCC 7120, which forms a terminally differentiated cell called a heterocyst with a semi-regular spacing of 10-15 cells. Because heterocysts are specialized cells for nitrogen fixation, the intracellular oxygen level of heterocysts is maintained very low even when adjacent cells perform oxygenic photosynthesis. Pyruvate decarboxylase of Zymomonas mobilis and alcohol dehydrogenase of Synechocystis sp. PCC 6803 were exclusively expressed in heterocysts. Ethanol production was concomitant with nitrogen fixation in genetically engineered Anabaena sp. PCC 7120. Engineering of carbon metabolism in heterocysts improved ethanol production, and strain ET14, with an extra copy of the invB gene expressed from a heterocyst-specific promoter, produced 130.9 mg L -1 of ethanol after 9 days. ET14 produced 1681.9 mg L -1 of ethanol by increasing the CO 2 supply. Ethanol production per heterocyst cell was approximately threefold higher than that per cell of unicellular cyanobacterium. This study demonstrates the potential of heterocysts for anaerobic production of biofuels and commodity chemicals under oxygenic photosynthetic conditions.

Rescue of cell death and inflammation of a mouse model of complex 1-mediated vision loss by repurposed drug molecules.

Science.gov (United States)

Yu, Alfred K; Datta, Sandipan; McMackin, Marissa Z; Cortopassi, Gino A

2017-12-15

Inherited mitochondrial optic neuropathies, such as Leber's hereditary optic neuropathy (LHON) and Autosomal dominant optic atrophy (ADOA) are caused by mutant mitochondrial proteins that lead to defects in mitochondrial complex 1-driven ATP synthesis, and cause specific retinal ganglion cell (RGC) loss. Complex 1 defects also occur in patients with primary open angle glaucoma (POAG), in which there is specific RGC loss. The treatment of mitochondrial optic neuropathy in the US is only supportive. The Ndufs4 knockout (Ndufs4 KO) mouse is a mitochondrial complex 1-deficient model that leads to RGC loss and rapid vision loss and allows for streamlined testing of potential therapeutics. Preceding RGC loss in the Ndufs4 KO is the loss of starburst amacrine cells, which may be an important target in the mechanism of complex 1-deficient vision loss. Papaverine and zolpidem were recently shown to be protective of bioenergetic loss in cell models of optic neuropathy. Treatment of Ndufs4 KO mice with papaverine, zolpidem, and rapamycin-suppressed inflammation, prevented cell death, and protected from vision loss. Thus, in the Ndufs4 KO mouse model of mitochondrial optic neuropathy, papaverine and zolpidem provided significant protection from multiple pathophysiological features, and as approved drugs in wide human use could be considered for the novel indication of human optic neuropathy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The production of nitric oxide in EL4 lymphoma cells overexpressing growth hormone.

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Arnold, Robyn E; Weigent, Douglas A

2003-01-01

Growth hormone (GH) is produced by immunocompetent cells and has been implicated in the regulation of a multiplicity of functions in the immune system involved in growth and activation. However, the actions of endogenous or lymphocyte GH and its contribution to immune reactivity when compared with those of serum or exogenous GH are still unclear. In the present study, we overexpressed lymphocyte GH in EL4 lymphoma cells, which lack the GH receptor (GHR), to determine the role of endogenous GH in nitric oxide (NO) production and response to genotoxic stress. Western blot analysis demonstrated that the levels of GH increased approximately 40% in cells overexpressing GH (GHo) when compared with cells with vector alone. The results also show a substantial increase in NO production in cells overexpressing GH that could be blocked by N(G)-monomethyl-L-arginine (L-NMMA), an L-arginine analogue that competitively inhibits all three isoforms of nitric oxide synthase (NOS). No evidence was obtained to support an increase in peroxynitrite in cells overexpressing GH. Overexpression of GH increased NOS activity, inducible nitric oxide synthase (iNOS) promoter activity, and iNOS protein expression, whereas endothelial nitric oxide synthase and neuronal nitric oxide synthase protein levels were essentially unchanged. In addition, cells overexpressing GH showed increased arginine transport ability and intracellular arginase activity when compared with control cells. GH overexpression appeared to protect cells from the toxic effects of the DNA alkylating agent methyl methanesulfonate. This possibility was suggested by maintenance of the mitochondrial transmembrane potential in cells overexpressing GH when compared with control cells that could be blocked by L-NMMA. Taken together, the data support the notion that lymphocyte GH, independently of the GH receptor, may play a key role in the survival of lymphocytes exposed to stressful stimuli via the production of NO.

Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell

KAUST Repository

Wang, Aijie

2011-03-01

Hydrogen gas production from cellulose was investigated using an integrated hydrogen production process consisting of a dark fermentation reactor and microbial fuel cells (MFCs) as power sources for a microbial electrolysis cell (MEC). Two MFCs (each 25mL) connected in series to an MEC (72mL) produced a maximum of 0.43V using fermentation effluent as a feed, achieving a hydrogen production rate from the MEC of 0.48m 3 H 2/m 3/d (based on the MEC volume), and a yield of 33.2mmol H 2/g COD removed in the MEC. The overall hydrogen production for the integrated system (fermentation, MFC and MEC) was increased by 41% compared with fermentation alone to 14.3mmol H 2/g cellulose, with a total hydrogen production rate of 0.24m 3 H 2/m 3/d and an overall energy recovery efficiency of 23% (based on cellulose removed) without the need for any external electrical energy input. © 2010 Elsevier Ltd.

Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell.

Science.gov (United States)

Wang, Aijie; Sun, Dan; Cao, Guangli; Wang, Haoyu; Ren, Nanqi; Wu, Wei-Min; Logan, Bruce E

2011-03-01

Hydrogen gas production from cellulose was investigated using an integrated hydrogen production process consisting of a dark fermentation reactor and microbial fuel cells (MFCs) as power sources for a microbial electrolysis cell (MEC). Two MFCs (each 25 mL) connected in series to an MEC (72 mL) produced a maximum of 0.43 V using fermentation effluent as a feed, achieving a hydrogen production rate from the MEC of 0.48 m(3) H(2)/m(3)/d (based on the MEC volume), and a yield of 33.2 mmol H(2)/g COD removed in the MEC. The overall hydrogen production for the integrated system (fermentation, MFC and MEC) was increased by 41% compared with fermentation alone to 14.3 mmol H(2)/g cellulose, with a total hydrogen production rate of 0.24 m(3) H(2)/m(3)/d and an overall energy recovery efficiency of 23% (based on cellulose removed) without the need for any external electrical energy input. Copyright © 2010 Elsevier Ltd. All rights reserved.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

Directory of Open Access Journals (Sweden)

Fukuda Shin

2009-11-01

Full Text Available Abstract Background Theca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK. Methods Ovarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells. Results Akt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca

Hydrogen production and purification for fuel cell applications

Science.gov (United States)

Chin, Soo Yin

The increased utilization of proton-exchange membrane (PEM) fuel cells as an alternative to internal combustion engines is expected to increase the demand for hydrogen, which is used as the energy source in these systems. Currently, production of hydrogen for fuel cells is primarily achieved via steam reforming, partial oxidation or autothermal reforming of natural gas, or steam reforming of methanol. However, in all of these processes CO is a by-product that must be subsequently removed due to its adverse effects on the Pt-based electrocatalysts of the PEM fuel cell. Our efforts have focused on production of CO-free hydrogen via catalytic decomposition of hydrocarbons and purification of H2 via the preferential oxidation of CO. The catalytic decomposition of hydrocarbons is an attractive alternative for the production of H2. Previous studies utilizing methane have shown that this approach can indeed produce CO-free hydrogen, with filamentous carbon formed as the by-product and deposited on the catalyst. We have further extended this approach to the decomposition of ethane. In addition to hydrogen and filamentous carbon however, methane is also formed in this case as a by-product. Studies conducted at different temperatures and space velocities suggest that hydrogen is the primary product while methane is formed in a secondary step. Ni/SiO2 catalysts are active for ethane decomposition at temperatures above 500°C. Although the yield of hydrogen increases with temperature, the catalyst deactivation rate also accelerates at higher temperatures. The preferential oxidation of CO is currently used for the purification of CO-contaminated hydrogen streams due to its efficiency and simplicity. Conventional Pt catalysts used for this reaction have been shown to effectively remove CO, but have limited selectivity (i.e., substantial amounts of H 2 also react with O2). Our work focused on alternative catalytic materials, such as Ru and bimetallic Ru-based catalysts (Pt-Ru, Ru

Localization of diacylglycerol lipase alpha and monoacylglycerol lipase during postnatal development of the rat retina

Directory of Open Access Journals (Sweden)

Bruno eCécyre

2014-12-01

Full Text Available In recent decades, there has been increased interest in the physiological roles of the endocannabinoid (eCB system and its receptors, the cannabinoid receptor types 1 (CB1R and 2 (CB2R. Exposure to cannabinoids during development results in neurofunctional alterations, which implies that the eCB system is involved in the developmental processes of the brain. Because of their lipophilic nature, eCBs are synthesized on demand and are not stored in vesicles. Consequently, the enzymes responsible for their synthesis and degradation are key regulators of their physiological actions. Therefore, knowing the localization of these enzymes during development is crucial for a better understanding of the role played by eCBs during the formation of the central nervous system.In this study, we investigated the developmental protein localization of the synthesizing and catabolic enzymes of the principal eCB, 2-arachidonoylglycerol (2-AG in the retinas of young and adult rats. The distribution of the enzymes responsible for the synthesis (DAGLα and the degradation (MAGL of 2-AG was determined for every retinal cell type from birth to adulthood. Our results indicate that DAGLα is present early in postnatal development. It is highly expressed in photoreceptor, horizontal, amacrine, and ganglion cells. MAGL appears later during the development of the retina and its presence is limited to amacrine and Müller cells. Overall, these results suggest that 2-AG is strongly present in early retinal development and might be involved in the regulation of the structural and functional maturation of the retina.

Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells.

Science.gov (United States)

Hussain, Althaf I; Cordeiro, Melissa; Sevilla, Elizabeth; Liu, Jonathan

2010-05-14

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced

Concise review: stem cell-based approaches to red blood cell production for transfusion.

Science.gov (United States)

Shah, Siddharth; Huang, Xiaosong; Cheng, Linzhao

2014-03-01

Blood transfusion is a common procedure in modern medicine, and it is practiced throughout the world; however, many countries report a less than sufficient blood supply. Even in developed countries where the supply is currently adequate, projected demographics predict an insufficient supply as early as 2050. The blood supply is also strained during occasional widespread disasters and crises. Transfusion of blood components such as red blood cells (RBCs), platelets, or neutrophils is increasingly used from the same blood unit for multiple purposes and to reduce alloimmune responses. Even for RBCs and platelets lacking nuclei and many antigenic cell-surface molecules, alloimmunity could occur, especially in patients with chronic transfusion requirements. Once alloimmunization occurs, such patients require RBCs from donors with a different blood group antigen combination, making it a challenge to find donors after every successive episode of alloimmunization. Alternative blood substitutes such as synthetic oxygen carriers have so far proven unsuccessful. In this review, we focus on current research and technologies that permit RBC production ex vivo from hematopoietic stem cells, pluripotent stem cells, and immortalized erythroid precursors.

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation.

Science.gov (United States)

Cuerquis, Jessica; Romieu-Mourez, Raphaëlle; François, Moïra; Routy, Jean-Pierre; Young, Yoon Kow; Zhao, Jing; Eliopoulos, Nicoletta

2014-02-01

Mesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α-pretreated human bone marrow-derived MSCs on resting or activated T cells. MSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed. Unprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions. Unprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Hydrogen Production Performance of a 10-Cell Planar Solid-Oxide Electrolysis Stack

International Nuclear Information System (INIS)

James O'Brien; Carl Stoots; Steve Herring; J. Hartvigsen

2005-01-01

An experimental study is under way to assess the performance of solid-oxide cells operating in the steam electrolysis mode for hydrogen production over a temperature range of 800 to 900 C. Results presented in this paper were obtained from a ten-cell planar electrolysis stack, with an active area of 64 cm2 per cell. The electrolysis cells are electrolyte supported, with scandia-stabilized zirconia electrolytes (∼140 (micro)m thick), nickel-cermet steam/hydrogen electrodes, and manganite air-side electrodes. The metallic interconnect plates are fabricated from ferritic stainless steel. The experiments were performed over a range of steam inlet mole fractions (0.1-0.6), gas flow rates (1000-4000 sccm), and current densities (0 to 0.38 A/cm2). Steam consumption rates associated with electrolysis were measured directly using inlet and outlet dewpoint instrumentation. Cell operating potentials and cell current were varied using a programmable power supply. Hydrogen production rates up to 100 Normal liters per hour were demonstrated. Values of area-specific resistance and stack internal temperatures are presented as a function of current density. Stack performance is shown to be dependent on inlet steam flow rate

Effects of amalgam corrosion products on human cells

Energy Technology Data Exchange (ETDEWEB)

Goldschmidt, P R; Cogen, R B; Taubman, S B [Departments of Periodontics and Pathology, University of Connecticut Health Center, Farmington, Connecticut, U.S.A.

1976-01-01

Using three independent criteria, we have found that 10/sup -4/,10/sup -6/M concentrations of ions presumably liberated from the corrosion of dental amalgam produce injurious effects on either human gingival fibroblasts or HeLa cells when the cells are grown in culture. Release of /sup 51/Cr and uptake of trypan blue dye were seen with 10/sup -5/M Hg/sup + +/ and Ag/sup +/. Inhibition of amino acid incorporation into protein-like material was seen with eluates of amalgam and with ionic solutions of most metals comprising dental amalgam. Stannous ion showed little if any cytotoxic potential. These results suggest that corrosion products of amalgam are capable of causing cellular injury or destruction.

Prospective of biodiesel production utilizing microalgae as the cell ...

African Journals Online (AJOL)

Microalgae are sunlight-driven miniature factories that convert atmospheric CO2 to polar and neutral lipids which after esterification can be utilized as an alternative source of petroleum. Further, other metabolic products such as bioethanol and biohydrogen produced by algal cells are also being considered for the same ...

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

Science.gov (United States)

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-02-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

Ikaros imposes a barrier to CD8+ T cell differentiation by restricting autocrine IL-2 production.

Science.gov (United States)

O'Brien, Shaun; Thomas, Rajan M; Wertheim, Gerald B; Zhang, Fuqin; Shen, Hao; Wells, Andrew D

2014-06-01

Naive CD4(+) T cells require signals from the TCR and CD28 to produce IL-2, expand, and differentiate. However, these same signals are not sufficient to induce autocrine IL-2 production by naive CD8(+) T cells, which require cytokines provided by other cell types to drive their differentiation. The basis for failed autocrine IL-2 production by activated CD8(+) cells is unclear. We find that Ikaros, a transcriptional repressor that silences IL-2 in anergic CD4(+) T cells, also restricts autocrine IL-2 production by CD8(+) T cells. We find that CD8(+) T cell activation in vitro in the absence of exogenous cytokines and CD4 help leads to marked induction of Ikaros, a known repressor of the Il2 gene. Naive murine CD8 T cells haplo-insufficient for Ikzf1 failed to upregulate Ikaros, produced autocrine IL-2, and differentiated in an IL-2-dependent manner into IFN-γ-producing CTLs in response to TCR/CD28 stimulation alone. Furthermore, Ikzf1 haplo-insufficient CD8(+) T cells were more effective at controlling Listeria infection and B16 melanoma growth in vivo, and they could provide help to neighboring, non-IL-2-producing cells to differentiate into IFN-γ-producing effectors. Therefore, by repressing autocrine IL-2 production, Ikaros ensures that naive CD8(+) T cells remain dependent on licensing by APCs and CD4(+) T cells, and it may therefore act as a cell-intrinsic safeguard against inappropriate CTL differentiation and immunopathology. Copyright © 2014 by The American Association of Immunologists, Inc.

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

Science.gov (United States)

Corrêa de Freitas, Marcela Cristina; Bomfim, Aline de Sousa; Mizukami, Amanda; Picanço-Castro, Virgínia; Swiech, Kamilla; Covas, Dimas Tadeu

2017-09-01

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 μg and 202.6 μg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture.

Science.gov (United States)

Ratcliffe, Elizabeth; Hourd, Paul; Guijarro-Leach, Juan; Rayment, Erin; Williams, David J; Thomas, Robert J

2013-01-01

Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes.

Laser processes and system technology for the production of high-efficient crystalline solar cells

Science.gov (United States)

Mayerhofer, R.; Hendel, R.; Zhu, Wenjie; Geiger, S.

2012-10-01

The laser as an industrial tool is an essential part of today's solar cell production. Due to the on-going efforts in the solar industry, to increase the cell efficiency, more and more laser-based processes, which have been discussed and tested at lab-scale for many years, are now being implemented in mass production lines. In order to cope with throughput requirements, standard laser concepts have to be improved continuously with respect to available average power levels, repetition rates or beam profile. Some of the laser concepts, that showed high potential in the past couple of years, will be substituted by other, more economic laser types. Furthermore, requirements for processing with less-heat affected zones fuel the development of industry-ready ultra short pulsed lasers with pulse widths even below the picosecond range. In 2011, the German Ministry of Education and Research (BMBF) had launched the program "PV-Innovation Alliance", with the aim to support the rapid transfer of high-efficiency processes out of development departments and research institutes into solar cell production lines. Here, lasers play an important role as production tools, allowing the fast implementation of high-performance solar cell concepts. We will report on the results achieved within the joint project FUTUREFAB, where efficiency optimization, throughput enhancement and cost reduction are the main goals. Here, the presentation will focus on laser processes like selective emitter doping and ablation of dielectric layers. An indispensable part of the efforts towards cost reduction in solar cell production is the improvement of wafer handling and throughput capabilities of the laser processing system. Therefore, the presentation will also elaborate on new developments in the design of complete production machines.

Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber.

Science.gov (United States)

Chen, Ke-Quan; Li, Jian; Ma, Jiang-Feng; Jiang, Min; Wei, Ping; Liu, Zhong-Min; Ying, Han-Jie

2011-01-01

The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Hyperglycosylated human chorionic gonadotropin does not increase progesterone production by luteinized granulosa cells.

Science.gov (United States)

Crochet, John R; Shah, Anish A; Schomberg, David W; Price, Thomas M

2012-09-01

Trophoblast-derived human chorionic gonadotropin (hCG) promotes corpus luteum progesterone (P4) production, and wide ranges of serum P4 levels are noted in various pregnancy outcomes, despite similar hCG concentrations. There are five unique biologically active hCG variants in human pregnancy urine, and previous studies of P4 production in response to hCG have used only preparations containing all isoforms. Understanding exactly which hCG variant is primarily responsible for stimulating corpus luteum steroidogenesis may have great clinical and diagnostic implications, including in the setting of ectopic pregnancy. Our objective was to delineate the role of the standard and hyperglycosylated (H)-hCG isoforms in stimulating P4 production by luteinized granulosa cells. Cell culture, ELISA, and fluorometric-based protein assays were done at Duke University Medical Center. Patients were anonymous oocyte donors. Cultured luteinized granulosa cells were treated with 0.25, 0.5, and 1.0 ng/ml total hCG, which contains all isoforms, purified standard hCG (37.1 kDa), and purified H-hCG (42.8 kDa). P4 produced per total cellular protein (nanograms per microgram) was measured via ELISA and fluorometric protein determination kits. Both total hCG (P = 0.0003) and purified standard hCG (P production. Purified H-hCG did not change the P4 produced per total cellular protein response (P value not significant). Standard hCG stimulated P4 production by cultured granulosa cells and likely supports corpus luteum function via interactions with the LH/hCG receptor. In contrast, H-hCG did not increase P4 production, which indicates a nonsteroidogenic role for this protein during early gestation.

Hydrothermal decomposition of yeast cells for production of proteins and amino acids

Energy Technology Data Exchange (ETDEWEB)

Lamoolphak, Wiwat [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Goto, Motonobu [Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 850-8555 (Japan); Sasaki, Mitsuru [Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 850-8555 (Japan); Suphantharika, Manop [Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400 (Thailand); Muangnapoh, Chirakarn [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Prommuag, Chattip [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Shotipruk, Artiwan [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand)]. E-mail: artiwan.s@chula.ac.th

2006-10-11

This study examines hydrothermal decomposition of Baker's yeast cells, used as a model for spent Brewer's yeast waste, into protein and amino acids. The reaction was carried out in a closed batch reactor at various temperatures between 100 and 250 deg. C. The reaction products were separated into water-soluble and solid residue. The results demonstrated that the amount of yeast residue decreased with increasing hydrolysis temperature. After 20 min reaction in water at 250 deg. C, 78% of yeast was decomposed. The highest amount of protein produced was also obtained at this condition and was found to be 0.16 mg/mg dry yeast. The highest amount of amino acids (0.063 mg/mg dry yeast) was found at the lowest temperature tested after 15 min. The hydrolysis product obtained at 200 deg. C was tested as a nutrient source for yeast growth. The growth of yeast cells in the culture medium containing 2 w/v% of this product was comparable to that of the cells grown in the medium containing commercial yeast extract at the same concentration. These results demonstrated the feasibility of using subcritical water to potentially decompose proteinaceous waste such as spent Brewer's yeast while recovering more useful products.

Hydrothermal decomposition of yeast cells for production of proteins and amino acids

International Nuclear Information System (INIS)

Lamoolphak, Wiwat; Goto, Motonobu; Sasaki, Mitsuru; Suphantharika, Manop; Muangnapoh, Chirakarn; Prommuag, Chattip; Shotipruk, Artiwan

2006-01-01

This study examines hydrothermal decomposition of Baker's yeast cells, used as a model for spent Brewer's yeast waste, into protein and amino acids. The reaction was carried out in a closed batch reactor at various temperatures between 100 and 250 deg. C. The reaction products were separated into water-soluble and solid residue. The results demonstrated that the amount of yeast residue decreased with increasing hydrolysis temperature. After 20 min reaction in water at 250 deg. C, 78% of yeast was decomposed. The highest amount of protein produced was also obtained at this condition and was found to be 0.16 mg/mg dry yeast. The highest amount of amino acids (0.063 mg/mg dry yeast) was found at the lowest temperature tested after 15 min. The hydrolysis product obtained at 200 deg. C was tested as a nutrient source for yeast growth. The growth of yeast cells in the culture medium containing 2 w/v% of this product was comparable to that of the cells grown in the medium containing commercial yeast extract at the same concentration. These results demonstrated the feasibility of using subcritical water to potentially decompose proteinaceous waste such as spent Brewer's yeast while recovering more useful products

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer.

Science.gov (United States)

Salehmin, M N I; Annuar, M S M; Chisti, Y

2013-11-01

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

Science.gov (United States)

Wertheimer, Tobias; Velardi, Enrico; Tsai, Jennifer; Cooper, Kirsten; Xiao, Shiyun; Kloss, Christopher C.; Ottmüller, Katja J.; Mokhtari, Zeinab; Brede, Christian; deRoos, Paul; Kinsella, Sinéad; Palikuqi, Brisa; Ginsberg, Michael; Young, Lauren F.; Kreines, Fabiana; Lieberman, Sophia R.; Lazrak, Amina; Guo, Peipei; Malard, Florent; Smith, Odette M.; Shono, Yusuke; Jenq, Robert R.; Hanash, Alan M.; Nolan, Daniel J.; Butler, Jason M.; Beilhack, Andreas; Manley, Nancy R.; Rafii, Shahin; Dudakov, Jarrod A; van den Brink, Marcel RM

2018-01-01

The thymus is extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood and this capacity diminishes considerably with age. Here we show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration, via their production of BMP4. ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signalling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance and regeneration; and its downstream targets such as Dll4, itself a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity. PMID:29330161

Electricity production from microbial fuel cell by using yeast

International Nuclear Information System (INIS)

Vorasingha, A.; Souvakon, C.; Boonchom, K.

2006-01-01

The continuous search for methods to generate electricity from renewable sources such as water, solar energy, wind, nuclear or chemicals was discussed with particular focus on attaining the full power of the microbial fuel cell (MFC). Under ideal environmental conditions, the only byproducts of a biofuel cell would be water and carbon dioxide (CO 2 ). The production of energy from renewables such as biomass is important for sustainable development and reducing global emissions of CO 2 . Hydrogen can also be an important component of an energy infrastructure that reduces CO 2 emissions if the hydrogen is produced from renewable sources and used in fuel cells. Hydrogen gas can be biologically produced at high concentration from the fermentation of high sugar substrates such as glucose and sucrose. Some of the issues of MFC design were addressed, including the use of cheap substrates to derive microbial electricity. In the MFC, yeast donates electrons to a chemical electron mediator, which in turn transfers the electrons to an electrode, producing electricity. Experimental results showed that glucose yielded the highest peak voltage, but a semi-processed sugar and molasses were similar to glucose in the electricity production pattern. It was noted that this technology is only at the research stages, and more research is needed before household microbial fuel cells can be made available for producing power for prolonged periods of time. Future research efforts will focus on increasing the efficiency, finding alternatives to hazardous electron mediators and finding new microbes. 12 refs., 6 figs

Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

Science.gov (United States)

Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

2016-01-01

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

Science.gov (United States)

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

Maturing of SOFC cell and stack production technology and preparation for demonstration of SOFC stacks. Part 2

Energy Technology Data Exchange (ETDEWEB)

2006-07-01

The TOFC/Riso pilot plant production facility for the manufacture of anode-supported cells has been further up-scaled with an automated continuous spraying process and an extra sintering capacity resulting in production capacity exceeding 15,000 standard cells (12x12 cm2) in 2006 with a success rate of about 85% in the cell production. All processing steps such as tape-casting, spraying, screen-printing and atmospheric air sintering in the cell production have been selected on condition that up-scaling and cost effective, flexible, industrial mass production are feasible. The standard cell size is currently being increased to 18x18 cm2, and 150 cells of this size have been produced in 2006 for our further stack development. To improve quality and lower production cost, a new screen printing line is under establishment. TOFC's stack design is an ultra compact multilayer assembly of cells (including contact layers), metallic interconnects, spacer frames and glass seals. The compactness ensures minimized material consumption and low cost. Standard stacks with cross flow configuration contains 75 cells (12x12cm2) delivering about 1.2 kW at optimal operation conditions with pre-reformed NG as fuel. Stable performance has been demonstrated for 500-1000 hours. Significantly improved materials, especially concerning the metallic interconnect and the coatings have been introduced during the last year. Small stacks (5-10 cells) exhibit no detectable stack degradation using our latest cells and stack materials during test periods of 500-1000 hours. Larger stacks (50-75 cells) suffer from mal-distribution of gas and air inside the stacks, gas leakage, gas cross-over, pressure drop, and a certain loss of internal electrical contact during operation cycles. Measures have been taken to find solutions during the following development work. The stack production facilities have been improved and up-scaled. In 2006, 5 standard stacks have been assembled and burned in based on

The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells

Directory of Open Access Journals (Sweden)

Vaclav Janovec

2018-02-01

Full Text Available Recent studies have reported that the crosslinking of regulatory receptors (RRs, such as blood dendritic cell antigen 2 (BDCA-2 (CD303 or ILT7 (CD85g, of plasmacytoid dendritic cells (pDCs efficiently suppresses the production of type I interferons (IFN-I, α/β/ω and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9 ligands. The exact mechanism of how this B cell receptor (BCR-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

Science.gov (United States)

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.