Silicon-on Ceramic Process: Silicon Sheet Growth and Device Development for the Large-area Silicon Sheet and Cell Development Tasks of the Low-cost Solar Array Project

NASA Technical Reports Server (NTRS)

Chapman, P. W.; Zook, J. D.; Heaps, J. D.; Grung, B. L.; Koepke, B.; Schuldt, S. B.

1979-01-01

The technical and economic feasibility of producing solar cell-quality silicon was investigated. This was done by coating one surface of carbonized ceramic substrates with a thin layer of large-grain polycrystalline silicon from the melt. Significant progress in the following areas was demonstrated: (1) fabricating a 10 sq cm cell having 9.9 percent conversion efficiency; (2) producing a 225 sq cm layer of sheet silicon; and (3) obtaining 100 microns thick coatings at pull speed of 0.15 cm/sec, although approximately 50 percent of the layer exhibited dendritic growth.

Paint it Black: One-Step Etch Cuts Solar Cell Costs - Continuum Magazine

Science.gov Websites

quicker, cheaper way to produce large volumes of high-performance PV devices. Cost is a major obstacle for propel PV toward cost-competitiveness. A New Approach to Antireflection Any light reflected from a solar etching large pyramids into the cell surface, add considerable cost to a solar cell-and they succeed only

Role of bone morphogenetic protein-7 in renal fibrosis

PubMed Central

Li, Rui Xi; Yiu, Wai Han; Tang, Sydney C. W.

2015-01-01

Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM) formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET). BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application. PMID:25954203

Multiplication of VHS virus in insect cells.

PubMed

Lorenzen, N; Olesen, N J

1995-01-01

Viral haemorrhagic septicaemia virus (VHSV) belongs to the rhabdovirus family and is a major pathogen in farmed rainbow trout. An insect cell culture traditionally used for production of recombinant proteins was found to be susceptible to VHS virus. At pH 6.2, VHSV multiplication induced formation of large syncytia similar to those obtained by baculovirus-induced expression of recombinant VHSV glycoprotein. The VHSV G protein produced in insect cells was smaller than G protein derived from fish cells. VHS virus produced in insect cells was still pathogenic to rainbow trout after 2 cell culture passages.

Large area, low cost space solar cells with optional wraparound contacts

NASA Technical Reports Server (NTRS)

Michaels, D.; Mendoza, N.; Williams, R.

1981-01-01

Design parameters for two large area, low cost solar cells are presented, and electron irradiation testing, thermal alpha testing, and cell processing are discussed. The devices are a 2 ohm-cm base resistivity silicon cell with an evaporated aluminum reflector produced in a dielectric wraparound cell, and a 10 ohm-cm silicon cell with the BSF/BSR combination and a conventional contact system. Both cells are 5.9 x 5.9 cm and require 200 micron thick silicon material due to mission weight constraints. Normalized values for open circuit voltage, short circuit current, and maximum power calculations derived from electron radiation testing are given. In addition, thermal alpha testing values of absorptivity and emittance are included. A pilot cell processing run produced cells averaging 14.4% efficiencies at AMO 28 C. Manufacturing for such cells will be on a mechanized process line, and the area of coverslide application technology must be considered in order to achieve cost effective production.

Inhibition of T Helper Cell Type 2 Cell Differentiation and Immunoglobulin E Response by Ligand-Activated Vα14 Natural Killer T Cells

PubMed Central

Cui, Junqing; Watanabe, Naohiro; Kawano, Tetsu; Yamashita, Masakatsu; Kamata, Tohru; Shimizu, Chiori; Kimura, Motoko; Shimizu, Eiko; Koike, Jyunzo; Koseki, Haruhiko; Tanaka, Yujiro; Taniguchi, Masaru; Nakayama, Toshinori

1999-01-01

Murine Vα14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Vα14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-γ upon in vivo stimulation with a specific ligand, α-galactosylceramide (α-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Vα14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Vα14 NKT-deficient mice that Vα14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with α-GalCer, Vα14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Vα14 NKT– or IFN-γ–deficient mice. Consistent with these results, we also found that ligand-activated Vα14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Vα14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-γ. In marked contrast, our studies have revealed that IL-4 produced by Vα14 NKT cells has only a minor effect on Th2 cell differentiation. PMID:10499917

Large area low-cost space solar cell development

NASA Technical Reports Server (NTRS)

Baraona, C. R.; Cioni, J. L.

1982-01-01

A development program to produce large-area (5.9 x 5.9 cm) space quality silicon solar cells with a cost goal of 30 $/watt is descibed. Five cell types under investigation include wraparound dielectric, mechanical wraparound and conventional contact configurations with combinations of 2 or 10 ohm-cm resistivity, back surface reflectors and/or fields, and diffused or ion implanted junctions. A single step process to cut cell and cover-glass simultaneously is being developed. A description of cell developments by Applied Solar Energy Corp., Spectrolab and Spire is included. Results are given for cell and array tests, performed by Lockheed, TRW and NASA. Future large solar arrays that might use cells of this type are discussed.

New bimetallic EMF cell shows promise in direct energy conversion

NASA Technical Reports Server (NTRS)

Hesson, J. C.; Shimotake, H.

1968-01-01

Concentration cell, based upon a thermally regenerative cell principle, produces electrical energy from any large heat source. This experimental bimetallic EMF cell uses a sodium-bismuth alloy cathode and a pure liquid sodium anode. The cell exhibits reliability, corrosion resistance, and high current density performance.

Size and competitive mating success in the yeast Saccharomyces cerevisiae.

PubMed

Smith, Carl; Pomiankowski, Andrew; Greig, Duncan

2014-03-01

In unicellular organisms like yeast, mating with the right partner is critical to future fitness because each individual can only mate once. Because cell size is important for viability, mating with a partner of the right size could be a significant advantage. To investigate this idea, we manipulated the size of unmated yeast cells and showed that their viability depended on environmental conditions; large cells do better on rich medium and small cells do better on poor medium. We also found that the fitness of offspring is determined by the size of their parents. Finally, we demonstrated that when a focal cell of one mating type was placed with a large and a small cell of the opposite mating type, it was more likely to mate with the cell that was closer to the optimum size for growth in a given environment. This pattern was not generated by differences in passive mating efficiency of large and small cells across environments but by competitive mating behavior, mate preference, or both. We conclude that the most likely mechanism underlying this interesting behavior is that yeast cells compete for mates by producing pheromone signals advertising their viability, and cells with the opportunity to choose prefer to mate with stronger signalers because such matings produce more viable offspring.

Epithelial stem cells are formed by small-particles released from particle-producing cells

PubMed Central

Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

2017-01-01

Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

Low-cost production of solar-cell panels

NASA Technical Reports Server (NTRS)

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.

Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

PubMed Central

Bäckström, Malin; Link, Thomas; Olson, Fredrik J; Karlsson, Hasse; Graham, Rosalind; Picco, Gianfranco; Burchell, Joy; Taylor-Papadimitriou, Joyce; Noll, Thomas; Hansson, Gunnar C

2003-01-01

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer. PMID:12950230

Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

DOEpatents

Dees, H. Craig

1998-01-01

Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

DOEpatents

Dees, H.C.

1998-05-26

Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

Kidney cell electrophoresis in space flight: Rationale, methods, results and flow cytometry applications

NASA Technical Reports Server (NTRS)

Todd, P.; Morrison, Dennis R.; Barlow, Grant H.; Lewis, Marian L.; Lanham, J. W.; Cleveland, C.; Williams, K.; Kunze, M. E.; Goolsby, C. L.

1988-01-01

Cultures of human embryonic kidney cells consistently contain an electrophoretically separable subpopulation of cells that produce high levels of urokinase and have an electrophoretic mobility about 85 percent as high as that of the most mobile human embryonic kidney cells. This subpopulation is rich in large epithelioid cells that have relatively little internal structure. When resolution and throughput are adequate, free fluid electrophoresis can be used to isolate a broad band of low mobility cells which also produces high levels of plasminogen activators (PAs). In the course of performing this, it was discovered that all electrophoretic subpopulations of cultured human embryonic kidney cells produce some PAs and that separate subpopulations produce high quantities of different types of PA's. This information and the development of sensitive assays for this project have provided new insights into cell secretion mechanisms related to fibrinolysis. These advances would probably not have been made without the NASA program to explore fundamental questions of free fluid electrophoresis in space.

Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

DOEpatents

Dees, H. Craig

1997-12-16

Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

NASA Astrophysics Data System (ADS)

Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

2005-05-01

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

Development of IL-22–producing NK lineage cells from umbilical cord blood hematopoietic stem cells in the absence of secondary lymphoid tissue

PubMed Central

Tang, Qin; Ahn, Yong-Oon; Southern, Peter; Blazar, Bruce R.; Miller, Jeffery S.

2011-01-01

Human secondary lymphoid tissues (SLTs) contain interleukin-22 (IL-22)–producing cells with an immature NK phenotype. Given their location, these cells are difficult to study. We have generated large numbers of NK22 cells from hematopoietic stem cells. HSC-derived NK22 cells show a CD56+CD117highCD94− phenotype, consistent with stage III NK progenitors. Like freshly isolated SLT stage III cells, HSC-derived NK22 cells express NKp44, CD161, CCR6, IL1 receptor, AHR, and ROR-γτ. IL-1β and IL-23 stimulation results in significant IL-22 but not interferon-γ production. Supernatant from these cells increases CD54 expression on mesenchymal stem cells. Thus, IL-22–producing NK cells can be generated in the absence of SLT. HSC-derived NK22 cells will be valuable in understanding this rare NK subset and create the opportunity for human translational clinical trials. PMID:21310921

Development of IL-22-producing NK lineage cells from umbilical cord blood hematopoietic stem cells in the absence of secondary lymphoid tissue.

PubMed

Tang, Qin; Ahn, Yong-Oon; Southern, Peter; Blazar, Bruce R; Miller, Jeffery S; Verneris, Michael R

2011-04-14

Human secondary lymphoid tissues (SLTs) contain interleukin-22 (IL-22)-producing cells with an immature NK phenotype. Given their location, these cells are difficult to study. We have generated large numbers of NK22 cells from hematopoietic stem cells. HSC-derived NK22 cells show a CD56(+)CD117(high)CD94(-) phenotype, consistent with stage III NK progenitors. Like freshly isolated SLT stage III cells, HSC-derived NK22 cells express NKp44, CD161, CCR6, IL1 receptor, AHR, and ROR-γτ. IL-1β and IL-23 stimulation results in significant IL-22 but not interferon-γ production. Supernatant from these cells increases CD54 expression on mesenchymal stem cells. Thus, IL-22-producing NK cells can be generated in the absence of SLT. HSC-derived NK22 cells will be valuable in understanding this rare NK subset and create the opportunity for human translational clinical trials.

Reassembly of Anterior Pituitary Organization by Hanging Drop Three-Dimensional Cell Culture

PubMed Central

Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components—collagens and laminin—were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization. PMID:24023396

Reassembly of anterior pituitary organization by hanging drop three-dimensional cell culture.

PubMed

Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2013-08-29

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.

Hollow microcarriers for large-scale expansion of anchorage-dependent cells in a stirred bioreactor.

PubMed

YekrangSafakar, Ashkan; Acun, Aylin; Choi, Jin-Woo; Song, Edward; Zorlutuna, Pinar; Park, Kidong

2018-03-26

With recent advances in biotechnology, mammalian cells are used in biopharmaceutical industries to produce valuable protein therapeutics and investigated as effective therapeutic agents to permanently degenerative diseases in cell based therapy. In these exciting and actively expanding fields, a reliable, efficient, and affordable platform to culture mammalian cells on a large scale is one of the most vital necessities. To produce and maintain a very large population of anchorage-dependent cells, a microcarrier-based stirred tank bioreactor is commonly used. In this approach, the cells are exposed to harmful hydrodynamic shear stress in the bioreactor and the mass transfer rates of nutrients and gases in the bioreactor are often kept below an optimal level to prevent cellular damages from the shear stress. In this paper, a hollow microcarrier (HMC) is presented as a novel solution to protect cells from shear stress in stirred bioreactors, while ensuring sufficient and uniform mass transfer rate of gases and nutrients. HMC is a hollow microsphere and cells are cultured on its inner surface to be protected, while openings on the HMC provide sufficient exchange of media inside the HMC. As a proof of concept, we demonstrated the expansion of fibroblasts, NIH/3T3 and the expansion and cardiac differentiation of human induced pluripotent stem cells, along with detailed numerical analysis. We believe that the developed HMC can be a practical solution to enable large-scale expansion of shear-sensitive anchorage-dependent cells in an industrial scale with stirred bioreactors. © 2018 Wiley Periodicals, Inc.

Effects of the oral administration of the exopolysaccharide produced by Lactobacillus kefiranofaciens on the gut mucosal immunity.

PubMed

Vinderola, Gabriel; Perdigón, Gabriela; Duarte, Jairo; Farnworth, Edward; Matar, Chantal

2006-12-01

The probiotic effects ascribed to lactic acid bacteria (LAB) and their fermented dairy products arise not only from whole microorganisms and cell wall components but also from peptides and extracellular polysaccharides (exopolysaccharides) produced during the fermentation of milk. There is a lack of knowledge concerning the immune mechanisms induced by exopolysaccharides produced by lactic acid bacteria, which would allow a better understanding of the functional effects described to them. The aim of this study was to investigate the in vivo immunomodulating capacity of the exopolysaccharide produced by Lactobacillus kefiranofaciens by analyzing the profile of cytokines and immunoglobulins induced at the intestinal mucosa level, in the intestinal fluid and blood serum. BALB/c mice received the exopolysaccharide produced by L. kefiranofaciens for 2, 5 or 7 consecutive days. At the end of each period of administration, control and treated mice were sacrificed and the numbers of IgA+ and IgG+ cells were determined on histological slices of the small and large intestine by immunofluorescence. Cytokines (IL-4, IL-6, IL-10, IL-12, IFNgamma and TNFalpha) were also determined in the gut lamina propria as well as in the intestinal fluid and blood serum. There was an increase of IgA+ cells in the small and large intestine lamina propria, without change in the number of IgG+ cells in the small intestine. This study reports the effects of the oral administration of the exopolysaccharide produced by L. kefiranofaciens in the number of IgA+ cells in the small and large intestine, comparing simultaneously the production of cytokines by cells of the lamina propria and in the intestinal fluid and blood serum. The increase in the number of IgA+ cells was not simultaneously accompanied by an enhance of the number of IL-4+ cells in the small intestine. This finding would be in accordance with the fact that, in general, polysaccharide antigens elicit a T-independent immune response. For IL-10+, IL-6+ and IL-12+ cells, the values found were slightly increased compared to control values, while IFNgamma+ and TNFalpha+ cells did not change compared to control values. The effects observed on immunoglobulins and in all the cytokines assayed in the large intestine after kefiran administration were of greater magnitude than the ones observed in the small intestine lamina propria, which may be due to the saccharolytic action of the colonic microflora. In the intestinal fluid, only IL-4 and IL-12 increased compared to control values. In blood serum, all the cytokines assayed followed a pattern of production quite similar to the one found for them in the small intestine lamina propria. We observed that the exopolysaccharide induced a gut mucosal response and it was able to up and down regulate it for protective immunity, maintaining intestinal homeostasis, enhancing the IgA production at both the small and large intestine level and influencing the systemic immunity through the cytokines released to the circulating blood.

Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators

PubMed Central

Coelho, Carolina; Sturny-Leclère, Aude; Fraser, James A.; Nielsen, Kirsten

2018-01-01

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to 100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways. PMID:29775480

Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

DOEpatents

Dees, H. Craig

1998-01-01

Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

DOEpatents

Dees, H.C.

1997-12-16

Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

PubMed Central

LAPTEVA, NATALIA; DURETT, APRIL G.; SUN, JIALI; ROLLINS, LISA A.; HUYE, LESLIE L.; FANG, JIAN; DANDEKAR, VARADA; MEI, ZHUYONG; JACKSON, KIMBERLEY; VERA, JUAN; ANDO, JUN; NGO, MINHTRAN C.; COUSTAN-SMITH, ELAINE; CAMPANA, DARIO; SZMANIA, SUSANN; GARG, TARUN; MORENO-BOST, AMBERLY; VANRHEE, FRITS; GEE, ADRIAN P.; ROONEY, CLIONA M.

2016-01-01

Background aims Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results Using this system we produced up to 19 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 CD3− CD56+ NK cells, within 8–10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8+ T cells within the NK cultures. However, these CD3+ T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy. PMID:22900959

Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

PubMed

Humbert, Olivier; Gisch, Don W; Wohlfahrt, Martin E; Adams, Amie B; Greenberg, Phil D; Schmitt, Tom M; Trobridge, Grant D; Kiem, Hans-Peter

2016-08-01

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

Origin of Matrix-Producing Cells That Contribute to Aortic Fibrosis in Hypertension.

PubMed

Wu, Jing; Montaniel, Kim Ramil C; Saleh, Mohamed A; Xiao, Liang; Chen, Wei; Owens, Gary K; Humphrey, Jay D; Majesky, Mark W; Paik, David T; Hatzopoulos, Antonis K; Madhur, Meena S; Harrison, David G

2016-02-01

Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1(+)) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1(+) progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1(+). Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I(+) cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1(+) progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix. © 2015 American Heart Association, Inc.

Exploring the cellular basis of human disease through a large-scale mapping of deleterious genes to cell types.

PubMed

Cornish, Alex J; Filippis, Ioannis; David, Alessia; Sternberg, Michael J E

2015-09-01

Each cell type found within the human body performs a diverse and unique set of functions, the disruption of which can lead to disease. However, there currently exists no systematic mapping between cell types and the diseases they can cause. In this study, we integrate protein-protein interaction data with high-quality cell-type-specific gene expression data from the FANTOM5 project to build the largest collection of cell-type-specific interactomes created to date. We develop a novel method, called gene set compactness (GSC), that contrasts the relative positions of disease-associated genes across 73 cell-type-specific interactomes to map genes associated with 196 diseases to the cell types they affect. We conduct text-mining of the PubMed database to produce an independent resource of disease-associated cell types, which we use to validate our method. The GSC method successfully identifies known disease-cell-type associations, as well as highlighting associations that warrant further study. This includes mast cells and multiple sclerosis, a cell population currently being targeted in a multiple sclerosis phase 2 clinical trial. Furthermore, we build a cell-type-based diseasome using the cell types identified as manifesting each disease, offering insight into diseases linked through etiology. The data set produced in this study represents the first large-scale mapping of diseases to the cell types in which they are manifested and will therefore be useful in the study of disease systems. Overall, we demonstrate that our approach links disease-associated genes to the phenotypes they produce, a key goal within systems medicine.

Novel Bioreactor Platform for Scalable Cardiomyogenic Differentiation from Pluripotent Stem Cell-Derived Embryoid Bodies.

PubMed

Rungarunlert, Sasitorn; Ferreira, Joao N; Dinnyes, Andras

2016-01-01

Generation of cardiomyocytes from pluripotent stem cells (PSCs) is a common and valuable approach to produce large amount of cells for various applications, including assays and models for drug development, cell-based therapies, and tissue engineering. All these applications would benefit from a reliable bioreactor-based methodology to consistently generate homogenous PSC-derived embryoid bodies (EBs) at a large scale, which can further undergo cardiomyogenic differentiation. The goal of this chapter is to describe a scalable method to consistently generate large amount of homogeneous and synchronized EBs from PSCs. This method utilizes a slow-turning lateral vessel bioreactor to direct the EB formation and their subsequent cardiomyogenic lineage differentiation.

Potential Polyunsaturated Aldehydes in the Strait of Gibraltar under Two Tidal Regimes

PubMed Central

Morillo-García, Soledad; Valcárcel-Pérez, Nerea; Cózar, Andrés; Ortega, María J.; Macías, Diego; Ramírez-Romero, Eduardo; García, Carlos M.; Echevarría, Fidel; Bartual, Ana

2014-01-01

Diatoms, a major component of the large-sized phytoplankton, are able to produce and release polyunsaturated aldehydes after cell disruption (potential PUAs or pPUA). These organisms are dominant in the large phytoplankton fraction (>10 µm) in the Strait of Gibraltar, the only connection between the Mediterranean Sea and the Atlantic Ocean. In this area, the hydrodynamics exerts a strong control on the composition and physiological state of the phytoplankton. This environment offers a great opportunity to analyze and compare the little known distribution of larger sized PUA producers in nature and, moreover, to study how environmental variables could affect the ranges and potential distribution of these compounds. Our results showed that, at both tidal regimes studied (Spring and Neap tides), diatoms in the Strait of Gibraltar are able to produce three aldehydes: Heptadienal, Octadienal and Decadienal, with a significant dominance of Decadienal production. The PUA released by mechanical cell disruption of large-sized collected cells (pPUA) ranged from 0.01 to 12.3 pmol from cells in 1 L, and from 0.1 to 9.8 fmol cell−1. Tidal regime affected the abundance, distribution and the level of physiological stress of diatoms in the Strait. During Spring tides, diatoms were more abundant, usually grouped nearer the coastal basin and showed less physiological stress than during Neap tides. Our results suggest a significant general increase in the pPUA productivity with increasing physiological stress for the cell also significantly associated to low nitrate availability. PMID:24633248

Cooperation of hTERT, SV40 T Antigen and Oncogenic Ras in Tumorigenesis: A Cell Transplantation Model Using Bovine Adrenocortical Cells1

PubMed Central

Thomas, Michael; Suwa, Tetsuya; Yang, Lianqing; Zhao, Lifang; Hawks, Christina L; Hornsby, Peter J

2002-01-01

Abstract Expression of TERT, the reverse transcriptase component of telomerase, is necessary to convert normal human cells to cancer cells. Despite this, “telomerization” by hTERT does not appear to alter the normal properties of cells. In a cell transplantation model in which bovine adrenocortical cells form vascularized tissue structures beneath the kidney capsule in scid mice, telomerization does not perturb the functional tissue-forming capacity of the cells. This cell transplantation model was used to study the cooperation of hTERT with SV40 T antigen (SV40 TAg) and oncogenic Ras in tumorigenesis. Only cells expressing all three genes were tumorigenic; this required large T, but not small t, antigen. These cells produced a continuously expanding tissue mass; they were invasive with respect to adjacent organs and eventually destroyed the kidney. Cells expressing only hTERT or only Ras produced minimally altered tissues. In contrast, SV40 TAg alone produced noninvasive nodules beneath the kidney capsule that had high proliferation rates balanced by high rates of apoptosis. The use of cell transplantation techniques in a cell type that is able to form tissue structures with or without full neoplastic conversion allows the phenotypes produced by individual cooperating oncogenes to be observed. PMID:12407443

Characterization of curdlan produced by Agrobacterium sp. IFO 13140 cells immobilized in a loofa sponge matrix, and application of this biopolymer in the development of functional yogurt.

PubMed

Ortiz Martinez, Camila; Pereira Ruiz, Suelen; Carvalho Fenelon, Vanderson; Rodrigues de Morais, Gutierrez; Luciano Baesso, Mauro; Matioli, Graciette

2016-05-01

Agrobacterium sp. IFO 13140 cells were immobilized on a loofa sponge and used to produce curdlan over five successive cycles. The interaction between microbial cells and the loofa sponge as well as the produced curdlan were characterized by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectrometry. The purity of the curdlan was also evaluated. The storage stability of the immobilized cells was assessed and the produced curdlan was used in a functional yogurt formulation. The average curdlan production by immobilized cells was 17.84 g L(-1) . The presence of the microorganism in the sponge was confirmed and did not cause alterations in the matrix, and the chemical structure of the curdlan was the same as that of commercial curdlan. The purity of both was similar. The immobilized cells remained active after 300 days of storage at -18 °C. The use of the produced curdlan in a functional yogurt resulted in a product with lower syneresis. A large number of cells physically adhered to the surface of loofa sponge fibers, and its use as an immobilization matrix to produce curdlan was effective. The use of the produced curdlan in yogurt allowed the development of a more stable product. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.

PubMed

Merten, Otto-Wilhelm; Charrier, Sabine; Laroudie, Nicolas; Fauchille, Sylvain; Dugué, Céline; Jenny, Christine; Audit, Muriel; Zanta-Boussif, Maria-Antonietta; Chautard, Hélène; Radrizzani, Marina; Vallanti, Giuliana; Naldini, Luigi; Noguiez-Hellin, Patricia; Galy, Anne

2011-03-01

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

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

Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko

We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventionalmore » DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.« less

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Complete in vitro oogenesis: retrospects and prospects.

PubMed

Wang, Jun-Jie; Ge, Wei; Liu, Jing-Cai; Klinger, Francesca Gioia; Dyce, Paul W; De Felici, Massimo; Shen, Wei

2017-11-01

Precise control of mammalian oogenesis has been a traditional focus of reproductive and developmental biology research. Recently, new reports have introduced the possibility of obtaining functional gametes derived in vitro from stem cells. The potential to produce functional gametes from stem cells has exciting applications for regenerative medicine though still remains challenging. In mammalian females ovulation and fertilization is a privilege reserved for a small number of oocytes. In reality the vast majority of oocytes formed from primordial germ cells (PGCs) will undergo apoptosis, or other forms of cell death. Removal occurs during germ cell cyst breakdown and the establishment of the primordial follicle (PF) pool, during the long dormancy at the PF stage, or through follicular atresia prior to reaching the ovulatory stage. A way to solve this limitation could be to produce large numbers of oocytes, in vitro, from stem cells. However, to recapitulate mammalian oogenesis and produce fertilizable oocytes in vitro is a complex process involving several different cell types, precise follicular cell-oocyte reciprocal interactions, a variety of nutrients and combinations of cytokines, and precise growth factors and hormones depending on the developmental stage. In 2016, two papers published by Morohaku et al. and Hikabe et al. reported in vitro procedures that appear to reproduce efficiently these conditions allowing for the production, completely in a dish, of a relatively large number of oocytes that are fertilizable and capable of giving rise to viable offspring in the mouse. The present article offers a critical overview of these results as well as other previous work performed mainly in mouse attempting to reproduce oogenesis completely in vitro and considers some perspectives for the potential to adapt the methods to produce functional human oocytes.

Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

DOEpatents

Dees, H.C.

1998-07-14

Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

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

PubMed

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.

Cell-free protein synthesis: applications in proteomics and biotechnology.

PubMed

He, Mingyue

2008-01-01

Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

PubMed

Conway, Michael K; Gerger, Michael J; Balay, Erin E; O'Connell, Rachel; Hanson, Seth; Daily, Neil J; Wakatsuki, Tetsuro

2015-05-14

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Mechanisms of cell killing by the new anti-cancer drug SR 4233

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

Wang, J.

SR 4233 (3-amino-1,2,4-benzotriazine, 1,4-dioxide) is a new potential anti-cancer drug which has a highly selective toxicity to hypoxic cells. This study investigated the mechanism of cell killing by this drug. Enzymatic studies have shown that SR 4233 is reductively metabolized to SR 4317 by the tumor cell lines SCVII and HT 1080 under hypoxic conditions. Cytochrome P-450 may play a major role in the reduction in both cell lines. DT diaphorase is the second most important enzyme in reducing SR 4233. In characterizing the major cellular target for SR 4233, the author has shown that damage to cell mitochondria ismore » produced largely under aerobic conditions, whereas DNA is likely to be the major target for cell death under hypoxic conditions. Further experiments demonstrated that DNA damage was similar to that produced by ionizing radiation at equitoxic doses, and chromosome aberrations can entirely account for cell death by SR 4233 under hypoxic conditions in the low dose range. Nevertheless, chromosome breaks produced by SR 4233 are less repairable than those produced by ionizing radiation, suggesting highly localized damage in the DNA by discrete foci of SR 4233 radicals.« less

Process in manufacturing high efficiency AlGaAs/GaAs solar cells by MO-CVD

NASA Technical Reports Server (NTRS)

Yeh, Y. C. M.; Chang, K. I.; Tandon, J.

1984-01-01

Manufacturing technology for mass producing high efficiency GaAs solar cells is discussed. A progress using a high throughput MO-CVD reactor to produce high efficiency GaAs solar cells is discussed. Thickness and doping concentration uniformity of metal oxide chemical vapor deposition (MO-CVD) GaAs and AlGaAs layer growth are discussed. In addition, new tooling designs are given which increase the throughput of solar cell processing. To date, 2cm x 2cm AlGaAs/GaAs solar cells with efficiency up to 16.5% were produced. In order to meet throughput goals for mass producing GaAs solar cells, a large MO-CVD system (Cambridge Instrument Model MR-200) with a susceptor which was initially capable of processing 20 wafers (up to 75 mm diameter) during a single growth run was installed. In the MR-200, the sequencing of the gases and the heating power are controlled by a microprocessor-based programmable control console. Hence, operator errors can be reduced, leading to a more reproducible production sequence.

Highly Efficient Large-Scale Lentiviral Vector Concentration by Tandem Tangential Flow Filtration

PubMed Central

Cooper, Aaron R.; Patel, Sanjeet; Senadheera, Shantha; Plath, Kathrin; Kohn, Donald B.; Hollis, Roger P.

2014-01-01

Large-scale lentiviral vector (LV) concentration can be inefficient and time consuming, often involving multiple rounds of filtration and centrifugation. This report describes a simpler method using two tangential flow filtration (TFF) steps to concentrate liter-scale volumes of LV supernatant, achieving in excess of 2000-fold concentration in less than 3 hours with very high recovery (>97%). Large volumes of LV supernatant can be produced easily through the use of multi-layer flasks, each having 1720 cm2 surface area and producing ~560 mL of supernatant per flask. Combining the use of such flasks and TFF greatly simplifies large-scale production of LV. As a demonstration, the method is used to produce a very high titer LV (>1010 TU/mL) and transduce primary human CD34+ hematopoietic stem/progenitor cells at high final vector concentrations with no overt toxicity. A complex LV (STEMCCA) for induced pluripotent stem cell generation is also concentrated from low initial titer and used to transduce and reprogram primary human fibroblasts with no overt toxicity. Additionally, a generalized and simple multiplexed real- time PCR assay is described for lentiviral vector titer and copy number determination. PMID:21784103

A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

PubMed Central

Dietmair, Stefanie; Hodson, Mark P.; Quek, Lake-Ee; Timmins, Nicholas E.; Gray, Peter; Nielsen, Lars K.

2012-01-01

Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly. PMID:22937046

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

PubMed

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-03-21

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

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

PubMed

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.

Enhanced Cultivation Of Stimulated Murine B Cells

NASA Technical Reports Server (NTRS)

Sammons, David W.

1994-01-01

Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales

PubMed Central

Negrete, Alejandro; Kotin, Robert M.

2007-01-01

The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g.≥1016 particles per production run, utilizes Baculovirus Expression Vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred-tank bioreactors are single-use, disposable bioreactors, e.g. Wave™. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave™ bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave™ bioreactors. Comparable yields of rAAV, ~2e+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration. Keywords: adeno-associated vectors; large-scale production; stirred tank bioreactor; wave bioreactor; gene therapy. PMID:17606302

Catalytic Metal Free Production of Large Cage Structure Carbon Particles: A Candidate for Hydrogen Storage

NASA Technical Reports Server (NTRS)

Kimura, Yuki; Nuth, Joseph A., III; Ferguson, Frank T.

2005-01-01

We will demonstrate that carbon particles consisting of large cages can be produced without catalytic metal. The carbon particles were produced in CO gas as well as by introduction of 5% methane gas into the CO gas. The gas-produced carbon particles were able to absorb approximately 16.2 wt% of hydrogen. This value is 2.5 times higher than the 6.5 wt% goal for the vehicular hydrogen storage proposed by the Department of Energy in the USA. Therefore, we believe that this carbon particle is an excellent candidate for hydrogen storage for fuel cells.

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

PubMed

Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

2011-01-01

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

Critical issues for engineering cord blood stem cells to produce insulin.

PubMed

Denner, Larry; Urban, Randall J

2008-09-01

The objectives of using cord blood stem cells for treating type 1 diabetes are simple in principle yet complex in biological and molecular mechanisms. These are defined by the complexity of the insulin-producing unit of the pancreas, the islet. Islets are composed of various cell types that arise from diverse lineages and communicate by hormones, growth factors and small-molecule mediators. These processes are regulated by integration of signal transduction pathways. While advances have been made to engineer umbilical cord blood stem cells to produce insulin, these studies only illuminate the potential of such cells to fulfil a necessary, but not sufficient, requirement for transplantation. The challenges ahead demand detailed understanding of molecular mechanisms to move from an opportunistic, phenotypic approach to transplantation and amelioration of blood glucose, to an orderly and logical approach to a biologically and medically meaningful solution. The issues include expansion to generate large numbers of cells, self-renewal to regulate the destiny of cord blood stem cells to repopulate the hematopoietic system, and multipotency of stem cells to generate the distinct cell types of an islet.

Dip coating process: Silicon sheet growth development for the large-area silicon sheet task of the low-cost silicon solar array project

NASA Technical Reports Server (NTRS)

Heaps, J. D.; Maciolek, R. B.; Zook, J. D.; Harrison, W. B.; Scott, M. W.; Hendrickson, G.; Wolner, H. A.; Nelson, L. D.; Schuller, T. L.; Peterson, A. A.

1976-01-01

The technical and economic feasibility of producing solar cell quality sheet silicon by dip-coating one surface of carbonized ceramic substrates with a thin layer of large grain polycrystalline silicon was investigated. The dip-coating methods studied were directed toward a minimum cost process with the ultimate objective of producing solar cells with a conversion efficiency of 10% or greater. The technique shows excellent promise for low cost, labor-saving, scale-up potentialities and would provide an end product of sheet silicon with a rigid and strong supportive backing. An experimental dip-coating facility was designed and constructed, several substrates were successfully dip-coated with areas as large as 25 sq cm and thicknesses of 12 micron to 250 micron. There appears to be no serious limitation on the area of a substrate that could be coated. Of the various substrate materials dip-coated, mullite appears to best satisfy the requirement of the program. An inexpensive process was developed for producing mullite in the desired geometry.

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

PubMed Central

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

2015-01-01

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

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

Factors influencing alternative splice site utilization in vivo.

PubMed Central

Fu, X Y; Manley, J L

1987-01-01

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images PMID:3029566

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

PubMed

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

Comparative performances of microbial capacitive deionization cell and microbial fuel cell fed with produced water from the Bakken shale.

PubMed

Shrestha, Namita; Chilkoor, Govinda; Wilder, Joseph; Ren, Zhiyong Jason; Gadhamshetty, Venkataramana

2018-06-01

This study evaluates and compares the performance of microbial fuel cells (MFCs) and microbial capacitive deionization cells (MCDCs) fed with wastewater produced from the Bakken shale. The produced water was characterized by high levels of dissolved solids and chemical oxygen demand (COD). Two-compartment MFCs and three-compartment MCDCs were evaluated under batch-fed mode using mixed microbial consortia in the anode, ferricyanide in the cathode, and produced water as the electrolyte in the anode and capacitive deionization units. COD removal in the MFCs was 88%, while that in the MCDCs was limited to 76%. The lower performance of the MCDCs was due to the large impedance (6600 Ω cm 2 ) compared with the MFCs (870 Ω cm 2 ). However, the MCDCs achieved two-fold higher removal of dissolved solids. Both the MFCs and MCDCs suffered from a higher impedance induced by fouling in the latter stages of the operation. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of Insulin-Immunoreactive Cells and Endocrine Cells Within the Duct System of the Adult Human Pancreas.

PubMed

Li, Rong; Zhang, Xiaoxi; Yu, Lan; Zou, Xia; Zhao, Hailu

2016-01-01

The adult pancreatic duct system accommodates endocrine cells that have the potential to produce insulin. Here we report the characterization and distribution of insulin-immunoreactive cells and endocrine cells within the ductal units of adult human pancreas. Sequential pancreas sections from 12 nondiabetic adults were stained with biomarkers of ductal epithelial cells (cytokeratin 19), acinar cells (amylase), endocrine cells (chromogranin A; neuron-specific enolase), islet hormones (insulin, glucagon, somatostatin, pancreatic polypeptide), cell proliferation (Ki-67), and neogenesis (CD29). The number of islet hormone-immunoreactive cells increased from large ducts to the terminal branches. The insulin-producing cells outnumbered endocrine cells reactive for glucagon, somatostatin, or pancreatic polypeptide. The proportions of insulin-immunoreactive count compared with local islets (100% as a baseline) were 1.5% for the main ducts, 7.2% for interlobular ducts, 24.8% for intralobular ducts, 67.9% for intercalated ducts, and 348.9% for centroacinar cells. Both Ki-67- and CD29-labeled cells were predominantly localized in the terminal branches around the islets. The terminal branches also showed cells coexpressing islet hormones and cytokeratin 19. The adult human pancreatic ducts showed islet hormone-producing cells. The insulin-reactive cells predominantly localized in terminal branches where they may retain potential capability for β-cell neogenesis.

Facile and Scalable Fabrication of Highly Efficient Lead Iodide Perovskite Thin-Film Solar Cells in Air Using Gas Pump Method.

PubMed

Ding, Bin; Gao, Lili; Liang, Lusheng; Chu, Qianqian; Song, Xiaoxuan; Li, Yan; Yang, Guanjun; Fan, Bin; Wang, Mingkui; Li, Chengxin; Li, Changjiu

2016-08-10

Control of the perovskite film formation process to produce high-quality organic-inorganic metal halide perovskite thin films with uniform morphology, high surface coverage, and minimum pinholes is of great importance to highly efficient solar cells. Herein, we report on large-area light-absorbing perovskite films fabrication with a new facile and scalable gas pump method. By decreasing the total pressure in the evaporation environment, the gas pump method can significantly enhance the solvent evaporation rate by 8 times faster and thereby produce an extremely dense, uniform, and full-coverage perovskite thin film. The resulting planar perovskite solar cells can achieve an impressive power conversion efficiency up to 19.00% with an average efficiency of 17.38 ± 0.70% for 32 devices with an area of 5 × 2 mm, 13.91% for devices with a large area up to 1.13 cm(2). The perovskite films can be easily fabricated in air conditions with a relative humidity of 45-55%, which definitely has a promising prospect in industrial application of large-area perovskite solar panels.

Preferential induction of Th17 cells in vitro and in vivo by Fucogalactan from Ganoderma lucidum (Reishi).

PubMed

Yoshida, Hideyuki; Suzuki, Mayu; Sakaguchi, Ryota; Tani, Ito; Kotani, Hitoshi; Shudo, Norimasa; Yoshimura, Akihiko

2012-05-25

The mushroom known as Reishi (Ganoderma lucidum) has been used as an herbal medicine for tumor treatment and immune system activation. Because its effects on the differentiation of effector T helper cells have not yet been fully understood, we investigated the effects of Reishi and those of its principal ingredient, β-glucan, on the activation of dendritic cells and the differentiation of Th17 cells. Reishi extracts as well as purified β-glucan (Curdran) activated DCs and caused them to produce large amounts of IL-23. β-glucan also enhanced and sustained the transcription of IL-23p19. The MEK-ERK signaling pathway positively regulates IL-23p19 transcription in β-glucan-stimulated DCs. In a mixed leukocyte reaction, Reishi-stimulated DCs preferentially induced Th17 cells. Furthermore, orally-administrated Reishi increased the percentages of Th17 cells and the transcription levels of antimicrobial peptides. Our results show that Reishi and β-glucan activate DCs to produce large amounts of IL-23, which induces Th17 differentiation both in vitro and in vivo. Copyright © 2012 Elsevier Inc. All rights reserved.

Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Antibacterial Compounds.

PubMed

Kobras, Carolin Martina; Mascher, Thorsten; Gebhard, Susanne

2017-01-01

Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, i.e., the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. We here describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances. The system is based on the well-characterized promoter P liaI , which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

GLUTAMATE NEUROTOXICITY IN THE DEVELOPING RAT COCHLEA IS ANTAGONIZED BY KUNURENIC ACID AND MK-801

EPA Science Inventory

Glutamate (GLU) is neurotoxic in the neonatal rat cochlea, producing hearing impairment which is largely due to the death of spiral ganglion cells, whereas the receptor hair cells are spared. endritic fibers of the spiral ganglion are post-synaptic to the primary afferent synapse...

Silicon-on-ceramic Process: Silicon Sheet Growth and Device Development for the Large-area Silicon Sheet and Cell Development Tasks of the Low-cost Solar Array Project

NASA Technical Reports Server (NTRS)

Chapman, P. W.; Zook, J. D.; Heaps, J. D.; Grung, B. L.; Koepke, B.; Schuldt, S. B.

1979-01-01

Significant progress is reported in fabricating a 4 sq cm cell having a 10.1 percent conversion efficiency and a 10 sq cm cell having a 9.2 percent conversion efficiency. The continuous (SCIM) coater succeeded in producing a 16 sq cm coating exhibiting unidirectional solidification and large grain size. A layer was grown at 0.2 cm/sec in the experimental coater which was partially dendritic but also contained a large smooth area approximately 100 micron m thick. The dark characteristic measurements of a typical SCC solar cell yield shunt resistance values of 10K ohms and series resistance values and 0.4 ohm. The production dip-coater is operating at over 50 percent yield in terms of good cell quality material. The most recent run yielded 13 good substrates out of 15.

Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis.

PubMed

Kawaguchi, Daichi; Furutachi, Shohei; Kawai, Hiroki; Hozumi, Katsuto; Gotoh, Yukiko

2013-01-01

Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone. Dll1 protein is induced in activated NSCs and segregates to one daughter cell during mitosis. Dll1-expressing cells reside in close proximity to quiescent NSCs, suggesting a feedback signal for NSC maintenance by their sister cells and progeny. Our data suggest a model in which NSCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis.

Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis

PubMed Central

Kawaguchi, Daichi; Furutachi, Shohei; Kawai, Hiroki; Hozumi, Katsuto; Gotoh, Yukiko

2013-01-01

Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone. Dll1 protein is induced in activated NSCs and segregates to one daughter cell during mitosis. Dll1-expressing cells reside in close proximity to quiescent NSCs, suggesting a feedback signal for NSC maintenance by their sister cells and progeny. Our data suggest a model in which NSCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis. PMID:23695674

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins

PubMed Central

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-01-01

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common. DOI: http://dx.doi.org/10.7554/eLife.24869.001 PMID:28323618

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

NASA Astrophysics Data System (ADS)

Zalzman, Michal; Gupta, Sanjeev; Giri, Ranjit K.; Berkovich, Irina; Sappal, Baljit S.; Karnieli, Ohad; Zern, Mark A.; Fleischer, Norman; Efrat, Shimon

2003-06-01

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade

PubMed Central

Choi, Jin Young; Kim, Seong Bum; Eo, Seong Kug

2015-01-01

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I–dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I–dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident–to-hematopoietic–to-resident cells that drives cytokine–to-chemokine–to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues. PMID:26618488

Biogas and Fuel Cells Workshop Summary Report: Proceedings from the Biogas and Fuel Cells Workshop, Golden, Colorado, June 11-13, 2012

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

Not Available

2013-01-01

The U.S. Department of Energy (DOE) National Renewable Energy Laboratory (NREL) held a Biogas and Fuel Cells Workshop June 11-13, 2012, in Golden, Colorado, to discuss biogas and waste-to-energy technologies for fuel cell applications. The overall objective was to identify opportunities for coupling renewable biomethane with highly efficient fuel cells to produce electricity; heat; combined heat and power (CHP); or combined heat, hydrogen and power (CHHP) for stationary or motive applications. The workshop focused on biogas sourced from wastewater treatment plants (WWTPs), landfills, and industrial facilities that generate or process large amounts of organic waste, including large biofuel production facilitiesmore » (biorefineries).« less

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

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

Wei, Pei; Li, Li; Qi, Hui

2012-02-10

Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas thatmore » expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.« less

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

PubMed Central

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

2014-01-01

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

Efficient production of erythroid, megakaryocytic and myeloid cells, using single cell-derived iPSC colony differentiation.

PubMed

Hansen, Marten; Varga, Eszter; Aarts, Cathelijn; Wust, Tatjana; Kuijpers, Taco; von Lindern, Marieke; van den Akker, Emile

2018-04-28

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications. Copyright © 2018. Published by Elsevier B.V.

Enhanced Oil Production by the Tropical Marine Diatom Thalassiosira Sp. Cultivated in Outdoor Photobioreactors.

PubMed

Kusumaningtyas, Pintaka; Nurbaiti, Santi; Suantika, Gede; Amran, Muhammad Bachri; Nurachman, Zeily

2017-08-01

Microalgae-derived oils have potential as a biofuel feedstock. To produce microalgal oils at a large scale, large amounts of nutrients and energy are needed to grow the algae. In this study, we evaluated three types of agricultural fertilizer (AF)-based culture media (AF1, AF2, and AF3) based on a previously published enriched seawater (ES) medium to produce biomass and oils from Thalassiosira sp. Under laboratory conditions, the highest cell productivity of Thalassiosira sp. was obtained with the AF3 medium. Thalassiosira sp. cultured in the AF3 medium produced 10.4 ± 0.9 mg L -1  day -1 oils, which is significantly higher than the 5.8 ± 0.7 mg L -1  day -1 produced in the ES medium. The higher production was due to the presence of nitrate and trace elements, both of which played roles in enhancing biomass and oil content, respectively. During cell growth, resting spores appeared inside the cells and were a marker to harvest the cells. Because of the abundant availability of sunlight in the tropics during the year, the oil production of Thalassiosira sp. in the AF3 medium was scaled up using outdoor photobioreactors under different weather conditions (rainy and dry seasons). Thalassiosira sp. produced more unsaturated fatty acids during the rainy season and produced more saturated fatty acids during the dry season. This study also demonstrated that it was possible to culture Thalassiosira sp. under outdoor conditions using a low-cost agricultural fertilizer-based culture medium (AF3 medium) to produce biodiesel feedstock with an annual production of 8.1 ± 0.4 t ha -1 during the dry season and of 23.9 ± 6.8 t ha -1 during the rainy season.

The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila.

PubMed

Li, Michelle A; Alls, Jeffrey D; Avancini, Rita M; Koo, Karen; Godt, Dorothea

2003-11-01

Interactions between somatic and germline cells are critical for the normal development of egg and sperm. Here we show that the gene traffic jam (tj) produces a soma-specific factor that controls gonad morphogenesis and is required for female and male fertility. tj encodes the only large Maf factor in Drosophila melanogaster, an orthologue of the atypical basic Leu zipper transcription factors c-Maf and MafB/Kreisler in vertebrates. Expression of tj occurs in somatic gonadal cells that are in direct contact with germline cells throughout development. In tj mutant gonads, somatic cells fail to inter-mingle and properly envelop germline cells, causing an early block in germ cell differentiation. In addition, tj mutant somatic cells show an increase in the level of expression for several adhesion molecules. We propose that tj is a critical modulator of the adhesive properties of somatic cells, facilitating germline-soma interactions that are essential for germ cell differentiation.

Reliable solution processed planar perovskite hybrid solar cells with large-area uniformity by chloroform soaking and spin rinsing induced surface precipitation

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

Chern, Yann-Cherng; Wu, Hung-Ruei; Chen, Yen-Chu

2015-08-15

A solvent soaking and rinsing method, in which the solvent was allowed to soak all over the surface followed by a spinning for solvent draining, was found to produce perovskite layers with high uniformity on a centimeter scale and with much improved reliability. Besides the enhanced crystallinity and surface morphology due to the rinsing induced surface precipitation that constrains the grain growth underneath in the precursor films, large-area uniformity with film thickness determined exclusively by the rotational speed of rinsing spinning for solvent draining was observed. With chloroform as rinsing solvent, highly uniform and mirror-like perovskite layers of area asmore » large as 8 cm × 8 cm were produced and highly uniform planar perovskite solar cells with power conversion efficiency of 10.6 ± 0.2% as well as much prolonged lifetime were obtained. The high uniformity and reliability observed with this solvent soaking and rinsing method were ascribed to the low viscosity of chloroform as well as its feasibility of mixing with the solvent used in the precursor solution. Moreover, since the surface precipitation forms before the solvent draining, this solvent soaking and rinsing method may be adapted to spinless process and be compatible with large-area and continuous production. With the large-area uniformity and reliability for the resultant perovskite layers, this chloroform soaking and rinsing approach may thus be promising for the mass production and commercialization of large-area perovskite solar cells.« less

Unique differentiation profile of mouse embryonic stem cells in rotary and stirred tank bioreactors.

PubMed

Fridley, Krista M; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B; Roy, Krishnendu

2010-11-01

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

A novel subset of helper T cells promotes immune responses by secreting GM-CSF

PubMed Central

Zhang, J; Roberts, A I; Liu, C; Ren, G; Xu, G; Zhang, L; Devadas, S; Shi, Yufang

2013-01-01

Helper T cells are crucial for maintaining proper immune responses. Yet, they have an undefined relationship with one of the most potent immune stimulatory cytokines, granulocyte macrophage-colony-stimulating factor (GM-CSF). By depleting major cytokines during the differentiation of CD4+ T cells in vitro, we derived cells that were found to produce large amounts of GM-CSF, but little of the cytokines produced by other helper T subsets. By their secretion of GM-CSF, this novel subset of helper T cells (which we have termed ThGM cells) promoted the production of cytokines by other T-cell subtypes, including type 1 helper T cell (Th1), type 2 helper T cell (Th2), type 1 cytotoxic T cell (Tc1), type 2 cytotoxic T cell (Tc2), and naive T cells, as evidenced by the fact that antibody neutralization of GM-CSF abolished this effect. ThGM cells were found to be highly prone to activation-induced cell death (AICD). Inhibitors of TRAIL or granzymes could not block AICD in ThGM cells, whereas inhibition of FasL/Fas interaction partially rescued ThGM cells from AICD. Thus, ThGM cells are a novel subpopulation of T helper cells that produce abundant GM-CSF, exhibit exquisite susceptibility to apoptosis, and therefore play a pivotal role in the regulation of the early stages of immune responses. PMID:24076588

Porcine platelet lysate as a supplement for animal cell culture

PubMed Central

Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

2007-01-01

A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (<10 EU/mL). The porcine platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

Scalable Production of Glioblastoma Tumor-initiating Cells in 3 Dimension Thermoreversible Hydrogels

NASA Astrophysics Data System (ADS)

Li, Qiang; Lin, Haishuang; Wang, Ou; Qiu, Xuefeng; Kidambi, Srivatsan; Deleyrolle, Loic P.; Reynolds, Brent A.; Lei, Yuguo

2016-08-01

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods, however, cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (~50 days, 10 passages tested, accumulative ~>1010-fold expansion) with both high growth rate (~20-fold expansion/7 days) and high volumetric yield (~2.0 × 107 cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient, affordable glioblastoma TICs for drug discovery.

Immunomodulatory Properties of Plants and Mushrooms.

PubMed

Martel, Jan; Ko, Yun-Fei; Ojcius, David M; Lu, Chia-Chen; Chang, Chih-Jung; Lin, Chuan-Sheng; Lai, Hsin-Chih; Young, John D

2017-11-01

Plants and mushrooms are used for medicinal purposes and the screening of molecules possessing biological activities. A single plant or mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on experimental conditions, but the reason behind this dichotomy remains obscure. We present here a large body of experimental data showing that water extracts of plants and mushrooms usually activate immune cells, whereas ethanol extracts inhibit immune cells. The mode of extraction of plants and mushrooms may thus determine the effects produced on immune cells, possibly due to differential solubility and potency of stimulatory and inhibitory compounds. We also examine the possibility of using such plant and mushroom extracts to treat immune system disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

PubMed

Tsai, Ang-Chen; Ma, Teng

2016-01-01

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

An Orthogonal and pH-Tunable Sensor-Selector for Muconic Acid Biosynthesis in Yeast.

PubMed

Snoek, Tim; Romero-Suarez, David; Zhang, Jie; Ambri, Francesca; Skjoedt, Mette L; Sudarsan, Suresh; Jensen, Michael K; Keasling, Jay D

2018-04-20

Microbes offer enormous potential for production of industrially relevant chemicals and therapeutics, yet the rapid identification of high-producing microbes from large genetic libraries is a major bottleneck in modern cell factory development. Here, we develop and apply a synthetic selection system in Saccharomyces cerevisiae that couples the concentration of muconic acid, a plastic precursor, to cell fitness by using the prokaryotic transcriptional regulator BenM driving an antibiotic resistance gene. We show that the sensor-selector does not affect production nor fitness, and find that tuning pH of the cultivation medium limits the rise of nonproducing cheaters. We apply the sensor-selector to selectively enrich for best-producing variants out of a large library of muconic acid production strains, and identify an isolate that produces more than 2 g/L muconic acid in a bioreactor. We expect that this sensor-selector can aid the development of other synthetic selection systems based on allosteric transcription factors.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

PubMed

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Microgravity

NASA Image and Video Library

2004-04-15

The manipulation of organic materials--cells, tissues, and even living organisms--offers many exciting possibilities for the future from organic computers to improved aquaculture. Commercial researchers are using the microgravity environment to produce large near perfect protein crystals Research on insulin has yielded crystals that far surpass the quality of insulin crystals grown on the ground. Using these crystals industry partners are working to develop new and improved treatments for diabetes. Other researchers are exploring the possibility of producing antibiotics using plant cell cultures which could lead to both orbital production and the improvement of ground-based antibiotic production.

Insulin

NASA Technical Reports Server (NTRS)

2004-01-01

The manipulation of organic materials--cells, tissues, and even living organisms--offers many exciting possibilities for the future from organic computers to improved aquaculture. Commercial researchers are using the microgravity environment to produce large near perfect protein crystals Research on insulin has yielded crystals that far surpass the quality of insulin crystals grown on the ground. Using these crystals industry partners are working to develop new and improved treatments for diabetes. Other researchers are exploring the possibility of producing antibiotics using plant cell cultures which could lead to both orbital production and the improvement of ground-based antibiotic production.

Titan Cells Confer Protection from Phagocytosis in Cryptococcus neoformans Infections

PubMed Central

Okagaki, Laura H.

2012-01-01

The human fungal pathogen Cryptococcus neoformans produces an enlarged “titan” cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells. PMID:22544904

Titan cells confer protection from phagocytosis in Cryptococcus neoformans infections.

PubMed

Okagaki, Laura H; Nielsen, Kirsten

2012-06-01

The human fungal pathogen Cryptococcus neoformans produces an enlarged "titan" cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells.

Polyploid titan cells produce haploid and aneuploid progeny to promote stress adaptation.

PubMed

Gerstein, Aleeza C; Fu, Man Shun; Mukaremera, Liliane; Li, Zhongming; Ormerod, Kate L; Fraser, James A; Berman, Judith; Nielsen, Kirsten

2015-10-13

Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans, yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions. The ability to adapt to stress is a key element for survival of pathogenic microbes in the host and thus plays an important role in pathogenesis. Here we investigated the predominantly haploid human fungal pathogen Cryptococcus neoformans, which is capable of ploidy and cell size increases during infection through production of titan cells. The enlarged polyploid titan cells are then able to rapidly undergo ploidy reduction to generate progeny with reduced ploidy and/or aneuploidy. Under stressful conditions, titan cell progeny have a growth and survival advantage over typical cell progeny. Understanding how titan cells enhance the rate of cryptococcal adaptation under stress conditions may assist in the development of novel drugs aimed at blocking ploidy transitions. Copyright © 2015 Gerstein et al.

Variability in the production of tannins and other polyphenols in cell cultures of 12 Nordic plant species.

PubMed

Suvanto, Jussi; Nohynek, Liisa; Seppänen-Laakso, Tuulikki; Rischer, Heiko; Salminen, Juha-Pekka; Puupponen-Pimiä, Riitta

2017-08-01

The polyphenol profiles of 18 cell cultures from 12 plant species were screened. The detected polyphenol fingerprints were diverse and differed from polyphenol profiles typically found in corresponding plant species. Cell cultures originating from 12 different plant species growing or grown in the Nordic countries were screened for their ability to synthesize polyphenols to assess their suitability for future studies and applications. The focus was on plant families Rosaceae and Ericaceae. On average, the Rosaceae cultures were the most efficient to produce hydrolysable tannins and the Ericaceae cultures were the most efficient to produce proanthocyanidins. This is in line with the general trend of polyphenols found in Rosaceae and Ericaceae leaves and fruits, even though several individual cell cultures differed from natural plants in their polyphenolic composition. Overall, several of the studied cell cultures exhibited capability in producing a large variety of polyphenols, including tannins with a high molecular weight, thus also showing promise for further studies concerning, for example, the accumulation of specific polyphenols or biosynthesis of polyphenols in the cell cultures.

Cell attachment functionality of bioactive conducting polymers for neural interfaces.

PubMed

Green, Rylie A; Lovell, Nigel H; Poole-Warren, Laura A

2009-08-01

Bioactive coatings for neural electrodes that are tailored for cell interactions have the potential to produce superior implants with improved charge transfer capabilities. In this study synthetically produced anionically modified laminin peptides DEDEDYFQRYLI and DCDPGYIGSR were used to dope poly(3,4-ethylenedioxythiophene) (PEDOT) electrodeposited on platinum (Pt) electrodes. Performance of peptide doped films was compared to conventional polymer PEDOT/paratoluene sulfonate (pTS) films using SEM, XPS, cyclic voltammetry, impedance spectroscopy, mechanical hardness and adherence. Bioactivity of incorporated peptides and their affect on cell growth was assessed using a PC12 neurite outgrowth assay. It was demonstrated that large peptide dopants produced softer PEDOT films with a minimal decrease in electrochemical stability, compared to the conventional dopant, pTS. Cell studies revealed that the YFQRYLI ligand retained neurite outgrowth bioactivity when DEDEDYFQRYLI was used as a dopant, but the effect was strongly dependant on initial cell attachment. Alternate peptide dopant, DCDPGYIGSR was found to impart superior cell attachment properties when compared to DEDEDYFQRYLI, but attachment on both peptide doped polymers could be enhanced by coating with whole native laminin.

Spraying Techniques for Large Scale Manufacturing of PEM-FC Electrodes

NASA Astrophysics Data System (ADS)

Hoffman, Casey J.

Fuel cells are highly efficient energy conversion devices that represent one part of the solution to the world's current energy crisis in the midst of global climate change. When supplied with the necessary reactant gasses, fuel cells produce only electricity, heat, and water. The fuel used, namely hydrogen, is available from many sources including natural gas and the electrolysis of water. If the electricity for electrolysis is generated by renewable energy (e.g., solar and wind power), fuel cells represent a completely 'green' method of producing electricity. The thought of being able to produce electricity to power homes, vehicles, and other portable or stationary equipment with essentially zero environmentally harmful emissions has been driving academic and industrial fuel cell research and development with the goal of successfully commercializing this technology. Unfortunately, fuel cells cannot achieve any appreciable market penetration at their current costs. The author's hypothesis is that: the development of automated, non-contact deposition methods for electrode manufacturing will improve performance and process flexibility, thereby helping to accelerate the commercialization of PEMFC technology. The overarching motivation for this research was to lower the cost of manufacturing fuel cell electrodes and bring the technology one step closer to commercial viability. The author has proven this hypothesis through a detailed study of two non-contact spraying methods. These scalable deposition systems were incorporated into an automated electrode manufacturing system that was designed and built by the author for this research. The electrode manufacturing techniques developed by the author have been shown to produce electrodes that outperform a common lab-scale contact method that was studied as a baseline, as well as several commercially available electrodes. In addition, these scalable, large scale electrode manufacturing processes developed by the author are also flexible and can be used to fabricate almost any fuel cell electrodes on the market today. This dissertation provides a description of the entire electrode manufacturing process as well as an analysis of the accuracy, performance and repeatability of the methods.

Detection of Oxytetracycline Production by Streptomyces rimosus in Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry

PubMed Central

Hansen, Lars Hestbjerg; Ferrari, Belinda; Sørensen, Anders Hay; Veal, Duncan; Sørensen, Søren Johannes

2001-01-01

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated Ptet promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil. PMID:11133451

Aircraft Fuel Cell Power Systems

NASA Technical Reports Server (NTRS)

Needham, Robert

2004-01-01

In recent years, fuel cells have been explored for use in aircraft. While the weight and size of fuel cells allows only the smallest of aircraft to use fuel cells for their primary engines, fuel cells have showed promise for use as auxiliary power units (APUs), which power aircraft accessories and serve as an electrical backup in case of an engine failure. Fuel cell MUS are both more efficient and emit fewer pollutants. However, sea-level fuel cells need modifications to be properly used in aircraft applications. At high altitudes, the ambient air has a much lower pressure than at sea level, which makes it much more difficult to get air into the fuel cell to react and produce electricity. Compressors can be used to pressurize the air, but this leads to added weight, volume, and power usage, all of which are undesirable things. Another problem is that fuel cells require hydrogen to create electricity, and ever since the Hindenburg burst into flames, aircraft carrying large quantities of hydrogen have not been in high demand. However, jet fuel is a hydrocarbon, so it is possible to reform it into hydrogen. Since jet fuel is already used to power conventional APUs, it is very convenient to use this to generate the hydrogen for fuel-cell-based APUs. Fuel cells also tend to get large and heavy when used for applications that require a large amount of power. Reducing the size and weight becomes especially beneficial when it comes to fuel cells for aircraft. My goal this summer is to work on several aspects of Aircraft Fuel Cell Power System project. My first goal is to perform checks on a newly built injector rig designed to test different catalysts to determine the best setup for reforming Jet-A fuel into hydrogen. These checks include testing various thermocouples, transmitters, and transducers, as well making sure that the rig was actually built to the design specifications. These checks will help to ensure that the rig will operate properly and give correct results when it is finally ready for testing. Another of my goals is to test new membranes for use in proton-exchange membrane fuel cells, in the hope that these membranes can increase the electricity that is produced by he1 cells. Producing more electricity means that fewer fuel cells are needed, thus reducing the weight and volume of an APU based on fuel cells, making such an APU much more viable.

[Histochemical study of the digestive organs of rats after a flight on "Kosmos-605"].

PubMed

Shubich, M G; Goriacheva, L L; Dudetskiĭ, V I; Lutsenko, N M; Mogil'naia, G M

1977-01-01

The histochemical study of the stomach, small and large intestines and pancreas of rats flown aboard the biosatellite Cosmos-605 as well as of synchronous and vivarium controls demonstrated a significant decline in the mucine producing capacity of epithelial cells of the stomach of the flight rats on the R + 1 day. The study showed an increased content of sialo- and sulphosaccharides in goblet cells of cryptae of large intestine and a reduced content of free cation protein in the acinar cells of the pancreas of flight rats. The changes were transient and disappeared by the R + 26 day.

ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

PubMed

Monath, Thomas P; Caldwell, Joseph R; Mundt, Wolfgang; Fusco, Joan; Johnson, Casey S; Buller, Mark; Liu, Jian; Gardner, Bridget; Downing, Greg; Blum, Paul S; Kemp, Tracy; Nichols, Richard; Weltzin, Richard

2004-10-01

The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.

Large Population-Based Study Reveals Disparities in Myeloma Precursor Disease | Center for Cancer Research

Cancer.gov

Multiple myeloma (MM) is a cancer of plasma cells, which are antibody-producing white blood cells. Patients with MM have a characteristic excess of monoclonal antibodies, so called M proteins, in their serum, urine, or both and plasma cell infiltration into their bone marrow at multiple sites. African Americans are more than twice as likely as whites to develop MM, but the

Materials for stem cell factories of the future

NASA Astrophysics Data System (ADS)

Celiz, Adam D.; Smith, James G. W.; Langer, Robert; Anderson, Daniel G.; Winkler, David A.; Barrett, David A.; Davies, Martyn C.; Young, Lorraine E.; Denning, Chris; Alexander, Morgan R.

2014-06-01

Polymeric substrates are being identified that could permit translation of human pluripotent stem cells from laboratory-based research to industrial-scale biomedicine. Well-defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large-scale drug-screening programs and clinical use. Yet more than 1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells is challenging, and a rethink of the current predominant cell-derived substrates is needed to provide technology that can be scaled to meet the needs of millions of patients a year. In this Review, we consider the role of materials discovery, an emerging area of materials chemistry that is in large part driven by the challenges posed by biologists to materials scientists.

Preliminary Engineering Study of Long-Lead Time Equipment Required for Large Lightweight Mirror Manufacture

DTIC Science & Technology

1981-06-01

numnber) Annealing Fusion Sealed Mirrors ULED Mirrors Boule Large Lightweight Mirror Core Low Expansion Glass Coremaker Mirror Blanks Forming Furnace...Experiments 34 4 10.6 Grinder Procurement 35 J 1 I GLOSSARY Alpha - Coef. of thermal expansion. Boule - The disc of glass formed in the furnace. Cell...turning over of large plates, cores or mirrors. Flowout - Method used to produce large diameter plates from small diameter boules. Glass - Used in the

Enhanced immune response of MAIT cells in tuberculous pleural effusions depends on cytokine signaling.

PubMed

Jiang, Jing; Chen, Xinchun; An, Hongjuan; Yang, Bingfen; Zhang, Fuping; Cheng, Xiaoxing

2016-09-02

The functions of MAIT cells at the site of Mycobacterium tuberculosis infection in humans are still largely unknown. In this study, the phenotypes and immune response of MAIT cells from tuberculous pleural effusions and peripheral blood were investigated. MAIT cells in tuberculous pleural effusions had greatly enhanced IFN-γ, IL-17F and granzyme B response compared with those in peripheral blood. The level of IFN-γ response in MAIT cells from tuberculous pleural effusions was inversely correlated with the extent of tuberculosis infection (p = 0.0006). To determine whether cytokines drive the immune responses of MAIT cells at the site of tuberculosis infection, the role of IL-1β, IL-2, IL-7, IL-12, IL-15 and IL-18 was investigated. Blockade of IL-2, IL-12 or IL-18 led to significantly reduced production of IFN-γ and/or granzyme B in MAIT cells from tuberculous pleural effusions. Majority of IL-2-producing cells (94.50%) in tuberculous pleural effusions had phenotype of CD3(+)CD4(+), and most IL-12p40-producing cells (91.39%) were CD14(+) cells. MAIT cells had significantly elevated expression of γc receptor which correlated with enhanced immune responses of MAIT cells. It is concluded that MAIT cells from tuberculous pleural effusions exhibited highly elevated immune response to Mtb antigens, which are controlled by cytokines produced by innate/adaptive immune cells.

Release of nitric oxide during the T cell-independent pathway of macrophage activation

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

Beckerman, K.P.; Rogers, H.W.; Corbett, J.A.

1993-02-01

Immunodeficient mice are remarkably resistant to Listeria monocytogenes (LM) infection. The authors examined the role that nitric oxide (NO) plays in the CB-17/lcr SCID (SCID) response to LM. SCID spleen cells produced large quantities of NO (as measured by nitrite formation) when incubated in the presence of heat-killed LM. NO produced large quantities of nitrite in response to LM, but only in the presence of IFN-[gamma]. The production of NO induced by LM was not affected by neutralizing antibodies to TNF or IL-1. The production of NO was inhibited by addition of either of two inhibitors of NO synthase, N[supmore » G]-monomethyl arginine, or aminoguanidine. In a different situation, NK cells that were stimulated by TNF and Listeria products to release IFN-[gamma] did not produce NO. Macrophages cultured with IFN-[gamma] killed live LM. This increased killing of LM was significantly inhibited by amino-guanidine. In vivo, administration of aminoguanidine resulted in a marked increase in the mortality and spleen bacterial loads of LM-infected SCID or immunocompetent control mice. It is concluded that NO is a critical effector molecule of T cell-independent natural resistence of LM as studied in the SCID mouse, and that the NO-mediated response is essential for both SCID and immunocompetent host to survive after LM infection. 37 refs., 7 figs.« less

The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus.

PubMed

Zhou, Jingxiang; Wang, Hao; Zhu, Xia; Li, Xingwei; Lv, Wenliang; Zhang, Dongming

2013-10-01

The explosive Koi herpesvirus (KHV) epidemic has caused the deaths of a large number of carp and carp variants and has produced serious economic losses. The mirror carp (Cyprinus carpio var. specularis) exhibits strong environmental adaptability and its primary cells can be used to isolate KHV. This study utilized the tissue explant method to systematically investigate primary cell culture conditions for mirror carp snout and caudal fin tissues. We demonstrated that cells from these two tissue types had strong adaptability, and when cultured in Medium 199 (M199) containing 20% serum at 26 to 30°C, the cells from the snout and caudal fin tissues exhibited the fastest egress and proliferation. Inoculation of these two cell types with KHV-infected fish kidney tissues produced typical cytopathic effects; additionally, identification by electron microscopy, and PCR indicated that KHV could be isolated from both cell types.

Liquid hyperpolarized 129Xe produced by phase exchange in a convection cell

NASA Astrophysics Data System (ADS)

Su, T.; Samuelson, G. L.; Morgan, S. W.; Laicher, G.; Saam, B.

2004-09-01

We present a method for the production of liquid hyperpolarized Xe129 that employs spin-exchange optical pumping in the gas phase and subsequent phase exchange with a column of xenon liquid. A convection loop inside the sealed glass cell allows efficient transfer of magnetization between the gas and liquid phases. By condensing to liquid a large fraction of the sample, this scheme permits the polarization of many more Xe129 atoms in a given sealed-cell volume than would otherwise be possible. We have thus far produced a steady-state polarization of 8% in 0.1mL of liquid with a characteristic rise time of ≈15min.

Solar Trees: First Large-Scale Demonstration of Fully Solution Coated, Semitransparent, Flexible Organic Photovoltaic Modules.

PubMed

Berny, Stephane; Blouin, Nicolas; Distler, Andreas; Egelhaaf, Hans-Joachim; Krompiec, Michal; Lohr, Andreas; Lozman, Owen R; Morse, Graham E; Nanson, Lana; Pron, Agnieszka; Sauermann, Tobias; Seidler, Nico; Tierney, Steve; Tiwana, Priti; Wagner, Michael; Wilson, Henry

2016-05-01

The technology behind a large area array of flexible solar cells with a unique design and semitransparent blue appearance is presented. These modules are implemented in a solar tree installation at the German pavilion in the EXPO2015 in Milan/IT. The modules show power conversion efficiencies of 4.5% and are produced exclusively using standard printing techniques for large-scale production.

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules.

PubMed

Verboogen, Daniëlle R J; Ter Beest, Martin; Honigmann, Alf; van den Bogaart, Geert

2018-05-01

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin-6 (IL-6) in human dendritic cells. By comparing this to the total secreted IL-6, we estimate that secretory vesicles contain about 0.5-3 IL-6 molecules, but with a large spread among cells/donors. Moreover, IL-6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL-6 production. IL-6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs. © 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Citizen journalism in a time of crisis: lessons from a large-scale California wildfire

Treesearch

S. Gillette; J. Taylor; D.J. Chavez; R. Hodgson; J. Downing

2007-01-01

The accessibility of news production tools through consumer communication technology has made it possible for media consumers to become media producers. The evolution of media consumer to media producer has important implications for the shape of public discourse during a time of crisis. Citizen journalists cover crisis events using camera cell phones and digital...

Genetic Engineering of Optical Properties of Biomaterials

NASA Astrophysics Data System (ADS)

Gourley, Paul; Naviaux, Robert; Yaffe, Michael

2008-03-01

Baker's yeast cells are easily cultured and can be manipulated genetically to produce large numbers of bioparticles (cells and mitochondria) with controllable size and optical properties. We have recently employed nanolaser spectroscopy to study the refractive index of individual cells and isolated mitochondria from two mutant strains. Results show that biomolecular changes induced by mutation can produce bioparticles with radical changes in refractive index. Wild-type mitochondria exhibit a distribution with a well-defined mean and small variance. In striking contrast, mitochondria from one mutant strain produced a histogram that is highly collapsed with a ten-fold decrease in the mean and standard deviation. In a second mutant strain we observed an opposite effect with the mean nearly unchanged but the variance increased nearly a thousand-fold. Both histograms could be self-consistently modeled with a single, log-normal distribution. The strains were further examined by 2-dimensional gel electrophoresis to measure changes in protein composition. All of these data show that genetic manipulation of cells represents a new approach to engineering optical properties of bioparticles.

FasL Mediates T-Cell Eradication of Tumor Cells Presenting Low Levels of Antigens | Center for Cancer Research

Cancer.gov

One approach to cancer immunotherapy, as opposed to therapeutic vaccination, is the transfusion of large numbers of tumor-specific killer T cells (cytotoxic T cells or CTLs) into patients. The body’s own defense killer T cells are a subgroup of T lymphocytes (a type of white blood cells) that are capable of inducing death in tumor cells. CTLs can cause the death of target cells either by releasing granules containing toxic molecules including perforin, or by producing a membrane protein called Fas ligand (FasL) which on interaction with the tumor cell results in cell death.

Formation of solid tumors by a single multinucleated cancer cel

PubMed Central

Weihua, Zhang; Lin, Qingtang; Ramoth, Asa J.; Fan, Dominic; Fidler, Isaiah J.

2011-01-01

BACKGROUND Large multinucleated cells (MNC) commonly exist in tumorigenic cancer cell lines widely used in research, but their contributions to tumorigenesis are unknown. METHODS In this study, we characterized MNCs in the murine fibrosarcoma cell line UV-2237 in vitro and in vivo at a single cell level. RESULTS We observed that MNCs originated from a rare subpopulation of mononuclear cells; MNCs were positive for a senescent marker, β-galacosidase (SA-β-Gal); MNCs were responsible for the majority of clonogenic activity when cultured in hard agar; MNCs were more resistant to chemotherapeutic agents than were mononuclear cells; MNCs could undergo asymmetric division (producing mononuclear cells) and self-renewal in vitro and in vivo; and, most importantly a single MNC produced orthotopic subcutaneous tumors (composed mainly of mononuclear cells) that gave rise to spontaneous lung metastases in nude mice. CONCLUSIONS MNCs can be growth-arrested under stress, are highly resistant to chemotherapy, and can generate clonal orthotopic metastatic tumors PMID:21365635

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

PubMed

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.

Clinical-scale expansion of mesenchymal stromal cells: a large banking experience.

PubMed

Lechanteur, Chantal; Briquet, Alexandra; Giet, Olivier; Delloye, Olivier; Baudoux, Etienne; Beguin, Yves

2016-05-20

Mesenchymal stromal cells (MSC) are largely investigated in clinical trials aiming to control inappropriate immune reactions (GVHD, Crohn's disease, solid organ transplantation). As the percentage of MSC precursors in bone marrow is very low, these must be expanded in vitro to obtain therapeutic cell doses. We describe here the constitution of an allogeneic human third-party MSC bank from screened healthy volunteer donors in compliance with quality specifications and ISCT-release criteria and report follow-up of different aspects of this activity since 2007. 68 clinical-grade large-scale MSC cultures were completed and analyzed. The whole process was described, including volunteer donor screening, bone marrow collection, mononuclear cell isolation and expansion over 4 weeks, harvesting, cryopreservation, release, administration and quality controls of the cells (including microbiology, phenotype, and potency assays). From 59 validated donors, 68 cultures were completed (mean of final yields: 886 × 10(6) cells/culture) and a total of 464 MSC aliquots have been produced and stored in liquid nitrogen (mean of 132.8 × 10(6) cells/bag). Each MSC batch underwent extensive testing to verify its conformity with EBMT and ISCT release criteria and was individually validated. As of June 1 2015, 314 bags have been released and infused to patients included in 6 different clinical protocols. All thawed MSC units satisfied to release criteria and no infusion-related toxicity was reported. In conclusion, despite low passage cultures, we have been able to create an allogeneic "off-the-shelf" MSC bank with a large number of frozen aliquots and report here an efficient clinical-grade MSC banking activity in place for more than 7 years. Our challenge now is to produce MSC in compliance with good manufacturing practices (GMP) as, in the meantime, MSC have become considered as advanced therapy medicinal products (ATMP). Another significant challenge remains the development of relevant potency assay.

Ultrasonic seam welding on thin silicon solar cells

NASA Technical Reports Server (NTRS)

Stofel, E. J.

1982-01-01

The ultrathin silicon solar cell has progressed to where it is a serious candidate for future light weight or radiation tolerant spacecraft. The ultrasonic method of producing welds was found to be satisfactory. These ultrathin cells could be handled without breakage in a semiautomated welding machine. This is a prototype of a machine capable of production rates sufficiently large to support spacecraft array assembly needs. For comparative purposes, this project also welded a variety of cells with thicknesses up to 0.23 mm as well as the 0.07 mm ultrathin cells. There was no electrical degradation in any cells. The mechanical pull strength of welds on the thick cells was excellent when using a large welding force. The mechanical strength of welds on thin cells was less since only a small welding force could be used without cracking these cells. Even so, the strength of welds on thin cells appears adequate for array application. The ability of such welds to survive multiyear, near Earth orbit thermal cycles needs to be demonstrated.

Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease

PubMed Central

Hickey, Raymond D.; Mao, Shennen A.; Glorioso, Jaime; Lillegard, Joseph B.; Fisher, James E.; Amiot, Bruce; Rinaldo, Piero; Harding, Cary O.; Marler, Ronald; Finegold, Milton J.; Grompe, Markus; Nyberg, Scott L.

2014-01-01

Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH+/− pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH−/− pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopthy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH−/− pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH−/− pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes. PMID:24879068

Cellulase producing microorganism ATCC 55702

DOEpatents

Dees, H. Craig

1997-01-01

Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Cellulase producing microorganism ATCC 55702

DOEpatents

Dees, H.C.

1997-12-30

Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

A Survey of Alternative Oxygen Production Technologies

NASA Technical Reports Server (NTRS)

Lueck, Dale E.; Parrish, Clyde F.; Buttner, William J.; Surma, Jan M.; Delgado, H. (Technical Monitor)

2000-01-01

Utilization of the Martian atmosphere for the production of fuel and oxygen has been extensively studied. The baseline fuel production process is a Sabatier reactor, which produces methane and water from carbon dioxide and hydrogen. The oxygen produced from the electrolysis of the water is only half of that needed for methane-based rocket propellant, and additional oxygen is needed for breathing air, fuel cells and other energy sources. Zirconia electrolysis cells for the direct reduction of CO2 are being developed as an alternative means of producing oxygen, but present many challenges for a large-scale oxygen production system. The very high operating temperatures and fragile nature of the cells coupled with fairly high operating voltages leave room for improvement. This paper will survey alternative oxygen production technologies, present data on operating characteristics, materials of construction, and some preliminary laboratory results on attempts to implement each.

Phylogenetic Analysis of the Synnema-Producing Genus Synnemapestaloides

PubMed Central

Watanabe, Kyoko; Sekiguchi, Mao; Sato, Toyozo; Hsiang, Tom; Kaneko, Shigeru; Tanaka, Kazuaki; Kanda, Masaru; Fujita, Naoko; Nozawa, Shunsuke

2016-01-01

Synnemapestaloides rhododendri, the type species of the genus Synnemapestaloides, is a pathogen of Rhododendron brachycarpum. This fungus produces six-celled conidia with appendages at both end cells, and are generated by annellidic conidiogenous cells on the synnema. These conidial structures are similar to those of the genus Pestalotia. The monotypic genus Synnemapestaloides is currently classified in the family Amphisphaeriaceae solely based on conidial morphology. Here we demonstrate that Synnemapestaloides represents a distinct genus in the family Sporocadaceae (Amphisphaeriales) based on differences in the nucleotide sequences of the partial large subunit rDNA gene, the rDNA internal transcribed spacer, and the partial β-tubulin. The genus most closely related to Synnemapestaloides is Seimatosporium and the species most similar to Synnemapestaloides rhododendri is Seim. foliicola which produces short synnema-like conidiomata (sporodochia). These results demonstrate that Seim. foliicola should be transferred to Synnemapestaloides, and also demonstrate that Sporocadaceae can have synnematal in addition to pycnidial and acervular conidiomata. PMID:29376945

Low cost silicon-on-ceramic photovoltaic solar cells

NASA Technical Reports Server (NTRS)

Koepke, B. G.; Heaps, J. D.; Grung, B. L.; Zook, J. D.; Sibold, J. D.; Leipold, M. H.

1980-01-01

A technique has been developed for coating low-cost mullite-based refractory substrates with thin layers of solar cell quality silicon. The technique involves first carbonizing one surface of the ceramic and then contacting it with molten silicon. The silicon wets the carbonized surface and, under the proper thermal conditions, solidifies as a large-grained sheet. Solar cells produced from this composite silicon-on-ceramic material have exhibited total area conversion efficiencies of ten percent.

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

NASA Technical Reports Server (NTRS)

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.

Fast Equalization for Large Lithium Ion Batteries

DTIC Science & Technology

2008-09-01

Lithium - ion batteries use an electrolyte that is flammable if exposed to high temperatures. Slight differences between the series-connected cells in a LiIon battery pack can produce imbalances in the cell voltages, and this greatly reduces the charge capacity. These batteries cannot be trickle charged like a lead acid battery because this would slightly overcharge some cells and would cause these cells to ignite. There are different methods used to ensure that the cells of a battery pack are not overcharged. The targeted equalizer (EQU) described here can

Neurokinin B-producing projection neurons in the lateral stripe of the striatum and cell clusters of the accumbens nucleus in the rat.

PubMed

Zhou, Ligang; Furuta, Takahiro; Kaneko, Takeshi

2004-12-06

Neurons producing preprotachykinin B (PPTB), the precursor of neurokinin B, constitute 5% of neurons in the dorsal striatum and project to the substantia innominata (SI) selectively. In the ventral striatum, PPTB-producing neurons are collected mainly in the lateral stripe of the striatum (LSS) and cell clusters of the accumbens nucleus (Acb). In the present study, we first examined the distribution of PPTB-immunoreactive neurons in rat ventral striatum and found that a large part of the PPTB-immunoreactive cell clusters was continuous to the LSS, but a smaller part was not. Thus, we divided the PPTB-immunoreactive cell clusters into the LSS-associated and non-LSS-associated ones. We next investigated the projection targets of the PPTB-producing ventral striatal neurons by combining immunofluorescence labeling and retrograde tracing. After injection of Fluoro-Gold into the basal component of the SI (SIb) and medial part of the interstitial nucleus of posterior limb of the anterior commissure, many PPTB-immunoreactive neurons were retrogradely labeled in the LSS-associated cell clusters and LSS, respectively. When the injection site included the ventral part of the sublenticular component of the SI(SIsl), retrogradely labeled neurons showed PPTB-immunoreactivity frequently in non-LSS-associated cell clusters. Furthermore, these PPTB-immunoreactive projections were confirmed by the double-fluorescence method after anterograde tracer injection into the ventral striatum containing the cell clusters. Since the dorsalmost part of the SIsl is known to receive strong inputs from PPTB-producing dorsal striatal neurons, the present results indicate that PPTB-producing ventral striatal neurons project to basal forebrain target regions in parallel with dorsal striatal neurons without significant convergence. 2004 Wiley-Liss, Inc.

Can a fermentation gas mainly produced by rumen Isotrichidae ciliates be a potential source of biohydrogen and a fuel for a chemical fuel cell?

PubMed

Piela, Piotr; Michałowski, Tadeusz; Miltko, Renata; Szewczyk, Krzysztof; Sikora, Radosław; Grzesiuk, Elzbieta; Sikora, Anna

2010-07-01

Bacteria, fungi and protozoa inhabiting the rumen, the largest chamber of the ruminants' stomach, release large quantities of hydrogen during the fermentation of carbohydrates. The hydrogen is used by coexisting methanogens to produce methane in energy-yielding processes. This work shows, for the first time, a fundamental possibility of using a hydrogen-rich fermentation gas produced by selected rumen ciliates to feed a low-temperature hydrogen fuel cell. A biohydrogen fuel cell (BHFC) was constructed consisting of (i) a bioreactor, in which a hydrogen-rich gas was produced from glucose by rumen ciliates, mainly of the Isotrichidae family, deprived of intra- and extracellular bacteria, methanogens, and fungi, and (ii) a chemical fuel cell of the polymer-electrolyte type (PEFC). The fuel cell was used as a tester of the technical applicability of the fermentation gas produced by the rumen ciliates for power generation. The average estimated hydrogen yield was ca. 1.15 mol H2 per mol of fermented glucose. The BHFC performance was equal to the performance of the PEFC running on pure hydrogen. No fuel cell poisoning effects were detected. A maximum power density of 1.66 kW/m2 (PEFC geometric area) was obtained at room temperature. The maximum volumetric power density was 128 W/m3 but the coulombic efficiency was only ca. 3.8%. The configuration of the bioreactor limited the continuous operation time of this BHFC to ca. 14 hours.

Activation of mouse liver natural killer cells and NK1.1(+) T cells by bacterial superantigen-primed Kupffer cells.

PubMed

Dobashi, H; Seki, S; Habu, Y; Ohkawa, T; Takeshita, S; Hiraide, H; Sekine, I

1999-08-01

Although bacterial superantigens have been well characterized as potent stimulators of T cells, their role in natural killer (NK)-type cells remains largely unknown. In the present study, we examined the effect of bacterial superantigens on mouse liver NK cells and NK1.1 Ag(+) (NK1(+)) T cells. C57BL/6 mice were intravenously injected with staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A), and mononuclear cells (MNC) of various organs were obtained from mice 4 hours after being injected with superantigen. MNC were cultured for 48 hours, and interferon gamma (IFN-gamma) levels of supernatants were measured. The antitumor cytotoxicities of the liver and spleen MNC were also evaluated 24 hours after the mice were injected with superantigen. Liver MNC produced more IFN-gamma than did splenocytes, and peripheral blood and lung MNC did not produce any detectable IFN-gamma. In addition, liver MNC acquired a potent antitumor cytotoxicity by the SEB injection, and both NK cells and NK1(+)T cells but not cluster of differentiation (CD)8(+) T cells were responsible for the cytotoxicity as demonstrated by either in vivo or in vitro cell depletion experiments, and the NK-type cells were partly responsible for the increased serum IFN-gamma. Activation of liver NK-type cells was also supported by the fact that liver NK cells proportionally increased and NK1(+) T cells augmented their CD11a expressions after SEB injection. The pretreatment of mice with anti-IFN-gamma Ab and/or with anti-interleukin-12 (IL-12) Ab diminished the SEB-induced cytotoxicity of liver MNC. Furthermore, the in vivo depletion of Kupffer cells decreased the SEB-induced cytotoxicity of liver MNC. Consistent with these results, liver MNC stimulated with superantigens in the presence of Kupffer cells in vitro produced a greater amount of IFN-gamma than did the liver MNC without Kupffer cells or splenocytes. Our results suggest that bacterial superantigen-primed Kupffer cells produce IL-12 and other monokines, while also nonspecifically activating both NK cells and NK1(+) T cells to produce IFN-gamma.

Heterogeneity in the growth hormone pituitary gland system of rats and humans: Implications to microgravity based research

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Grindeland, R.; Hayes, C.; Lanham, J. W.; Cleveland, C.; Todd, P.; Morrison, Dennis R.

1988-01-01

The cell separation techniques of velocity sedimentation, flow cytometry and continuous flow electrophoresis were used to obtain enriched populations of growth hormone (GH) cells. The goal was to isolate a GH cell subpopulation which releases GH molecules which are very high in biological activity, it was important to use a method which was effective in processing large numbers of cells over a short time span. The techniques based on sedimentation are limited by cell density overlaps and streaming. While flow cytometry is useful in the analytical mode for objectively establishing cell purity, the numbers of cells which can be processed in the sort mode are so small as to make this approach ineffective in terms of the long term goals. It was shown that continuous flow electrophoresis systems (CFES) can separate GH cells from other cell types on the basis of differences in surface charge. The bioreactive producers appear to be more electrophoretically mobile than the low producers. Current ground based CFES efforts are hampered by cell clumping in low ionic strength buffers and poor cell recoveries from the CFES device.

Optical volume and mass measurements show that mammalian cells swell during mitosis

PubMed Central

Zlotek-Zlotkiewicz, Ewa; Monnier, Sylvain; Cappello, Giovanni; Le Berre, Mael

2015-01-01

The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells. PMID:26598614

Detection of IgA antibodies and quantification of IgA antibody-producing cells specific for ovalbumin or Trichinella spiralis in the rat.

PubMed

Van Loveren, H; Osterhaus, A D; Nagel, J; Schuurman, H J; Vos, J G

1988-09-01

This report describes procedures to quantify IgA responses in the rat sensitized to ovalbumin or infected with the parasite Trichinella spiralis: an ELISPOT detecting specific IgA antibody-producing cells in lymph nodes, and an ELISA demonstrating IgA antibody in serum and gut mucosal scrapings. For this purpose a mouse monoclonal anti-rat IgA antibody was produced. This IgG1-kappa 1 antibody recognized rat IgA but not rat IgM, IgG, or IgE. It proved very suitable in both assays. Using this reagent we could demonstrate large numbers of IgA anti-ovalbumin-producing cells in the mesenteric lymph nodes 15 days after sensitization to ovalbumin via the Peyer's patches. At 28 days after sensitization the numbers were much lower. IgA antibody titres to ovalbumin in serum were maximal between days 14 and 21 after immunization. Maximal numbers of IgA anti-T. spiralis-producing cells were found in the mesenteric lymph nodes 12 days after infection with muscle larvae, followed by a sharp decrease at 15 days. Maximal IgA anti-T. spiralis antibody titres in serum and mucus scrapings of small intestines were found on days 10 and 12 after oral infection with the parasite.

Analysis of the genome of the sexually transmitted insect virus Hz-2V

USDA-ARS?s Scientific Manuscript database

Hz-2V is an insect DNA virus closely related to the baculoviruses that grow to high titers in insect cells and produces high yields of virus progeny. The capacity of this virus to replicate to high titers in insect cells may allow the use of this virus for production of large amount of proteins. Th...

A Murine Xenograft Model for Human CD30+ Anaplastic Large Cell Lymphoma

PubMed Central

Pfeifer, Walther; Levi, Edi; Petrogiannis-Haliotis, Tina; Lehmann, Leslie; Wang, Zhenxi; Kadin, Marshall E.

1999-01-01

To develop a model for the biology and treatment of CD30+ anaplastic large cell lymphoma (ALCL), we transplanted leukemic tumor cells from a 22-month-old girl with multiple relapsed ALCL. Tumor cells were inoculated intraperitoneally into a 4-week-old SCID/bg mouse and produced a disseminated tumor within 8 weeks; this tumor was serially transplanted by subcutaneous injections to other mice. Morphology, immunohistochemistry, and molecular genetics which demonstrated the NPM-ALK fusion protein, resulting from the t(2;5)(p23;q35), confirmed the identity of the xenograft with the original tumor. The tumor produced transcripts for interleukin-1α, tumor necrosis factor-α, and interferon-γ which could explain the patient’s B-symptoms. Treatment of mice with monoclonal antibody (HeFi-1) which activates CD30 antigen administered on day 1 after tumor transplantation prevented tumor growth. Treatment with HeFi-1 after tumors had reached a 0.2 cm3 volume caused tumor growth arrest and prevention of tumor dissemination. We conclude that transplantation of CD30+ ALCL to SCID/bg mice may provide a valuable model for the study of the biology and design of treatment modalities for CD30+ ALCL. PMID:10514417

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

PubMed Central

Brunstein, Maia; Teremetz, Maxime; Hérault, Karine; Tourain, Christophe; Oheim, Martin

2014-01-01

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2). PMID:24606927

Naive T-cell receptor transgenic T cells help memory B cells produce antibody

PubMed Central

Duffy, Darragh; Yang, Chun-Ping; Heath, Andrew; Garside, Paul; Bell, Eric B

2006-01-01

Injection of the same antigen following primary immunization induces a classic secondary response characterized by a large quantity of high-affinity antibody of an immunoglobulin G class produced more rapidly than in the initial response – the products of memory B cells are qualitatively distinct from that of the original naive B lymphocytes. Very little is known of the help provided by the CD4 T cells that stimulate memory B cells. Using antigen-specific T-cell receptor transgenic CD4 T cells (DO11.10) as a source of help, we found that naive transgenic T cells stimulated memory B cells almost as well (in terms of quantity and speed) as transgenic T cells that had been recently primed. There was a direct correlation between serum antibody levels and the number of naive transgenic T cells transferred. Using T cells from transgenic interleukin-2-deficient mice we showed that interleukin-2 was not required for a secondary response, although it was necessary for a primary response. The results suggested that the signals delivered by CD4 T cells and required by memory B cells for their activation were common to both antigen-primed and naive CD4 T cells. PMID:17067314

LentiPro26: novel stable cell lines for constitutive lentiviral vector production.

PubMed

Tomás, H A; Rodrigues, A F; Carrondo, M J T; Coroadinha, A S

2018-03-27

Lentiviral vectors (LVs) are excellent tools to promote gene transfer and stable gene expression. Their potential has been already demonstrated in gene therapy clinical trials for the treatment of diverse disorders. For large scale LV production, a stable producer system is desirable since it allows scalable and cost-effective viral productions, with increased reproducibility and safety. However, the development of stable systems has been challenging and time-consuming, being the selection of cells presenting high expression levels of Gag-Pro-Pol polyprotein and the cytotoxicity associated with some viral components, the main limitations. Hereby is described the establishment of a new LV producer cell line using a mutated less active viral protease to overcome potential cytotoxic limitations. The stable transfection of bicistronic expression cassettes with re-initiation of the translation mechanism enabled the generation of LentiPro26 packaging populations supporting high titers. Additionally, by skipping intermediate clone screening steps and performing only one final clone screening, it was possible to save time and generate LentiPro26-A59 cell line, that constitutively produces titers above 10 6 TU.mL -1 .day -1 , in less than six months. This work constitutes a step forward towards the development of improved LV producer cell lines, aiming to efficiently supply the clinical expanding gene therapy applications.

Silicon on ceramic process. Silicon sheet growth development for the large-area silicon sheet task of the low-cost silicon solar array project

NASA Technical Reports Server (NTRS)

Zook, J. D.; Heaps, J. D.; Maciolek, R. B.; Koepke, B. G.; Butter, C. D.; Schuldt, S. B.

1977-01-01

The technical and economic feasibility of producing solar-cell-quality sheet silicon was investigated. The sheets were made by coating one surface of carbonized ceramic substrates with a thin layer of large-grain polycrystalline silicon from the melt. Significant progress was made in all areas of the program.

Insect cells as factories for biomanufacturing.

PubMed

Drugmand, Jean-Christophe; Schneider, Yves-Jacques; Agathos, Spiros N

2012-01-01

Insect cells (IC) and particularly lepidopteran cells are an attractive alternative to mammalian cells for biomanufacturing. Insect cell culture, coupled with the lytic expression capacity of baculovirus expression vector systems (BEVS), constitutes a powerful platform, IC-BEVS, for the abundant and versatile formation of heterologous gene products, including proteins, vaccines and vectors for gene therapy. Such products can be manufactured on a large scale thanks to the development of efficient and scaleable production processes involving the integration of a cell growth stage and a stage of cell infection with the recombinant baculovirus vector. Insect cells can produce multimeric proteins functionally equivalent to the natural ones and engineered vectors can be used for efficient expression. Insect cells can be cultivated easily in serum- and protein-free media. A growing number of companies are currently developing an interest in producing therapeutics using IC-BEVS, and many products are today in clinical trials and on the market for veterinary and human applications. This review summarizes current knowledge on insect cell metabolism, culture conditions and applications. Copyright © 2011 Elsevier Inc. All rights reserved.

[The extraneuronal cholinergic system of the skin. Basic facts and clinical relevance].

PubMed

Kurzen, H

2004-05-01

Acetylcholine (ACh) is a prototypical neurotransmitter that has recently been recognized to occur extraneuronally in a large variety of cells. ACh and its nicotinic and muscarinic receptors are produced in the epidermis and in the adnexal structures of the skin in a highly complicated pattern. They are also produced in melanocytes, fibroblasts, endothelial cells and immune cells. Through autocrine, paracrine and endocrine mechanisms, the cholinergic system is involved in the basic functions of the skin, such as keratinocyte differentiation, epidermal barrier formation, sweating, sebum production, blood circulation, angiogenesis and a variety of immune reactions. Hence diseases like acne vulgaris, vitiligo, psoriasis, pemphigus vulgaris and atopic dermatitis may be influenced. The exploration of the extraneuronal cholinergic system of the skin has only just begun.

Enhanced immune response of MAIT cells in tuberculous pleural effusions depends on cytokine signaling

PubMed Central

Jiang, Jing; Chen, Xinchun; An, Hongjuan; Yang, Bingfen; Zhang, Fuping; Cheng, Xiaoxing

2016-01-01

The functions of MAIT cells at the site of Mycobacterium tuberculosis infection in humans are still largely unknown. In this study, the phenotypes and immune response of MAIT cells from tuberculous pleural effusions and peripheral blood were investigated. MAIT cells in tuberculous pleural effusions had greatly enhanced IFN-γ, IL-17F and granzyme B response compared with those in peripheral blood. The level of IFN-γ response in MAIT cells from tuberculous pleural effusions was inversely correlated with the extent of tuberculosis infection (p = 0.0006). To determine whether cytokines drive the immune responses of MAIT cells at the site of tuberculosis infection, the role of IL-1β, IL-2, IL-7, IL-12, IL-15 and IL-18 was investigated. Blockade of IL-2, IL-12 or IL-18 led to significantly reduced production of IFN-γ and/or granzyme B in MAIT cells from tuberculous pleural effusions. Majority of IL-2-producing cells (94.50%) in tuberculous pleural effusions had phenotype of CD3+CD4+, and most IL-12p40-producing cells (91.39%) were CD14+ cells. MAIT cells had significantly elevated expression of γc receptor which correlated with enhanced immune responses of MAIT cells. It is concluded that MAIT cells from tuberculous pleural effusions exhibited highly elevated immune response to Mtb antigens, which are controlled by cytokines produced by innate/adaptive immune cells. PMID:27586092

Molecular Cloning of Ghrelin and Characteristics of Ghrelin-Producing Cells in the Gastrointestinal Tract of the Common Marmoset (Callithrix jacchus).

PubMed

Takemi, Shota; Sakata, Ichiro; Apu, Auvijit Saha; Tsukahara, Shinji; Yahashi, Satowa; Katsuura, Goro; Iwashige, Fumihiro; Akune, Atsushi; Inui, Akio; Sakai, Takafumi

2016-10-01

Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.

The Echinoid Mitotic Gradient: Effect of Cell Size on the Micromere Cleavage Cycle

PubMed Central

Langelan Duncan, Rosalie E.; Whiteley, Arthur H.

2012-01-01

SUMMARY Like other euechinoids, the fertilized eggs of the sand dollar Dendraster excentricus proceed through cleavages that produce a pattern of macromeres, mesomeres, and micromeres at the 4th division. The 8 cells of the macro-mesomere lineage proceed through 6 additional cleavages before hatching. At the fifth overall division, the 4 micromeres produce a lineage of large micromeres that will divide 3 additional times, and a lineage of small micromeres that will divide once more before hatching. Irrespective of lineage, the length of the cell cycles is closely related to the size of the blastomere; cells of the same size have the same cell cycle time. A consequence is that at the fourth cleavage, there is a gradient of mitotic activity from the fastest dividers at the animal pole and the slowest cleacing micromeres at the vegetal pole. By the time of hatching, which is the 10th division of meso-macromeres, all cells are the same small size, the metachronic pattern of division gives way to asynchrony, and the mitotic gradient along the polar axis is lost. Experimental pre-exposure to sodium dodecyl sulfate (SDS), however, blocks the appearance of the gradients in cell size, the mitotic gradient, and the differential in cell cycle times. It is proposed that the mitotic gradients, cell cycle times, and attainment of a state of asynchrony are functions of cell size. Developmental consequences of the transition are large, and include coordinated activation of transcriptions, synthesis of new patterns of proteins, alterations of metabolism, and onset of morphogenesis. PMID:22006441

Multifaceted Activity of Listeriolysin O, the Cholesterol-Dependent Cytolysin of Listeria monocytogenes

PubMed Central

2014-01-01

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by numerous Gram-positive bacterial pathogens. These toxins are released in the extracellular environment as water-soluble monomers or dimers that bind to cholesterol-rich membranes and assemble into large pore complexes. Depending upon their concentration, the nature of the host cell and membrane (cytoplasmic or intracellular) they target, the CDCs can elicit many different cellular responses. Among the CDCs, listeriolysin O (LLO), which is a major virulence factor of the facultative intracellular pathogen Listeria monocytogenes, is involved in several stages of the intracellular lifecycle of the bacterium and displays unique characteristics. It has long been known that following L. monocytogenes internalization into host cells, LLO disrupts the internalization vacuole, enabling the bacterium to replicate into the host cell cytosol. LLO is then used by cytosolic bacteria to spread from cell to cell, avoiding bacterial exposure to the extracellular environment. Although LLO is continuously produced during the intracellular lifecycle of L. monocytogenes, several processes limit its toxicity to ensure the survival of infected cells. It was previously thought that LLO activity was limited to mediating vacuolar escape during bacterial entry and cell to cell spreading. This concept has been challenged by compelling evidence suggesting that LLO secreted by extracellular L. monocytogenes perforates the host cell plasma membrane, triggering important host cell responses. This chapter provides an overview of the well-established intracellular activity of LLO and the multiple roles attributed to LLO secreted by extracellular L. monocytogenes. PMID:24798012

Regulatory T cells in cattle and their potential role in bovine paratuberculosis.

PubMed

Coussens, Paul M; Sipkovsky, Sue; Murphy, Brooke; Roussey, Jon; Colvin, Christopher J

2012-05-01

The intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in wild and domestic ruminants. Johne's disease presents as a chronic enteritis with severe inflammation of intestinal tissues, characterized by widespread infiltration of macrophages, the target cell of MAP. Clinical signs of Johne's disease are typically accompanied by a loss of peripheral CD4+ T cell responses to MAP antigens and an increase in anti-MAP serum IgG levels. Recently, it was proposed that regulatory T cells might develop over the lengthy course of subclinical MAP infection. In the past five years, significant progress in defining bovine regulatory T cells has been made. These studies grew out of observations that IL-10 is produced by PBMCs in response to MAP antigen stimulation and that neutralization of this IL-10 could enhance IFN-γ production from MAP-antigen reactive effector T cells. Depletion studies revealed that MAP responsive cell populations producing IL-10 were largely CD4+ and CD25+, although monocytes have also been shown to produce IL-10 in response to MAP. In addition, evidence for a regulatory population of γδ T cells has also begun to accumulate. We summarize current thinking regarding regulatory T cells in MAP infection and provide data suggesting a potential link between regulatory T cells, bovine leukemia virus, and MAP. Copyright © 2012 Elsevier Ltd. All rights reserved.

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

PubMed Central

Throm, Robert E.; Ouma, Annastasia A.; Zhou, Sheng; Chandrasekaran, Anantharaman; Lockey, Timothy; Greene, Michael; De Ravin, Suk See; Moayeri, Morvarid; Malech, Harry L.; Sorrentino, Brian P.

2009-01-01

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1). PMID:19286997

Low-Temperature Soft-Cover Deposition of Uniform Large-Scale Perovskite Films for High-Performance Solar Cells.

PubMed

Ye, Fei; Tang, Wentao; Xie, Fengxian; Yin, Maoshu; He, Jinjin; Wang, Yanbo; Chen, Han; Qiang, Yinghuai; Yang, Xudong; Han, Liyuan

2017-09-01

Large-scale high-quality perovskite thin films are crucial to produce high-performance perovskite solar cells. However, for perovskite films fabricated by solvent-rich processes, film uniformity can be prevented by convection during thermal evaporation of the solvent. Here, a scalable low-temperature soft-cover deposition (LT-SCD) method is presented, where the thermal convection-induced defects in perovskite films are eliminated through a strategy of surface tension relaxation. Compact, homogeneous, and convection-induced-defects-free perovskite films are obtained on an area of 12 cm 2 , which enables a power conversion efficiency (PCE) of 15.5% on a solar cell with an area of 5 cm 2 . This is the highest efficiency at this large cell area. A PCE of 15.3% is also obtained on a flexible perovskite solar cell deposited on the polyethylene terephthalate substrate owing to the advantage of presented low-temperature processing. Hence, the present LT-SCD technology provides a new non-spin-coating route to the deposition of large-area uniform perovskite films for both rigid and flexible perovskite devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automation of large scale transient protein expression in mammalian cells

PubMed Central

Zhao, Yuguang; Bishop, Benjamin; Clay, Jordan E.; Lu, Weixian; Jones, Margaret; Daenke, Susan; Siebold, Christian; Stuart, David I.; Yvonne Jones, E.; Radu Aricescu, A.

2011-01-01

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. PMID:21571074

Realization of Quasi-Omnidirectional Solar Cells with Superior Electrical Performance by All-Solution-Processed Si Nanopyramids.

PubMed

Zhong, Sihua; Wang, Wenjie; Tan, Miao; Zhuang, Yufeng; Shen, Wenzhong

2017-11-01

Large-scale (156 mm × 156 mm) quasi-omnidirectional solar cells are successfully realized and featured by keeping high cell performance over broad incident angles (θ), via employing Si nanopyramids (SiNPs) as surface texture. SiNPs are produced by the proposed metal-assisted alkaline etching method, which is an all-solution-processed method and highly simple together with cost-effective. Interestingly, compared to the conventional Si micropyramids (SiMPs)-textured solar cells, the SiNPs-textured solar cells possess lower carrier recombination and thus superior electrical performances, showing notable distinctions from other Si nanostructures-textured solar cells. Furthermore, SiNPs-textured solar cells have very little drop of quantum efficiency with increasing θ, demonstrating the quasi-omnidirectional characteristic. As an overall result, both the SiNPs-textured homojunction and heterojunction solar cells possess higher daily electric energy production with a maximum relative enhancement approaching 2.5%, when compared to their SiMPs-textured counterparts. The quasi-omnidirectional solar cell opens a new opportunity for photovoltaics to produce more electric energy with a low cost.

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

PubMed

Volovitz, Ilan; Shapira, Netanel; Ezer, Haim; Gafni, Aviv; Lustgarten, Merav; Alter, Tal; Ben-Horin, Idan; Barzilai, Ori; Shahar, Tal; Kanner, Andrew; Fried, Itzhak; Veshchev, Igor; Grossman, Rachel; Ram, Zvi

2016-06-01

Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

Surgery-derived reactive oxygen species produced by polymorphonuclear leukocytes promote tumor recurrence: studies in an in vitro model.

PubMed

van Grevenstein, Wilhelmina M U; Aalbers, Arend G J; Ten Raa, Sander; Sluiter, Wim; Hofland, Leo J; Jeekel, Hans; van Eijck, Casper H J

2007-06-01

Tissue injury induces the acute phase response, aimed at minimizing damage and starting the healing process. Polymorphonuclear leukocytes (PMNs) respond to the presence of specific chemoattractants and begin to appear in large numbers. The aim of this study was to investigate the influence of reactive oxygen species (ROS) produced by PMNs on the interaction between colon carcinoma cells and mesothelial cells. An experimental human in vitro model was designed using Caco-2 colon carcinoma cells and primary cultures of mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with stimulated PMNs and unstimulated PMNs. Mesothelial cells were also incubated with xanthine/xanthine oxidase (X/XO) complex producing ROS after which adhesion of Caco-2 cells was investigated and the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) by means of enzyme immunoassay. In the control situation the average adhesion of Caco-2 cells to the mesothelial monolayers was 23%. Mesothelial monolayers incubated with unstimulated PMNs showed a 25% increase of tumor cell adhesion (P < 0.05). The adhesion of tumor to the monolayers incubated with the N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs increased with 40% (P < 0.01). Incubation of the mesothelium with X/XO resulted in an enhancement of adhesion of Caco-2 cells of 70% and an up-regulation of expression of ICAM-1, VCAM-1, and CD44. This study reveals an increase of tumor cell adhesion to the mesothelium induced by incubating the mesothelial monolayers with PMNs. PMNs are producing a number of products, like proteolytic enzymes, cytokines, and ROS. These factors up-regulate the expression of adhesion molecules and in that way stimulate the adhesion of tumor to the mesothelium.

Recognition of Glioma Stem Cells by Genetically Modified T Cells Targeting EGFRvIII and Development of Adoptive Cell Therapy for Glioma

PubMed Central

Johnson, Laura A.; Davis, Jeremy L.; Zheng, Zhili; Woolard, Kevin D.; Reap, Elizabeth A.; Feldman, Steven A.; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A.; Rosenberg, Steven A.

2012-01-01

Abstract No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application. PMID:22780919

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

PubMed

Morgan, Richard A; Johnson, Laura A; Davis, Jeremy L; Zheng, Zhili; Woolard, Kevin D; Reap, Elizabeth A; Feldman, Steven A; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A; Rosenberg, Steven A

2012-10-01

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

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

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Morphological alterations of T24 cells on flat and nanotubular TiO2 surfaces.

PubMed

Imani, Roghayeh; Kabaso, Doron; Erdani Kreft, Mateja; Gongadze, Ekaterina; Penic, Samo; Elersic, Kristina; Kos, Andrej; Veranic, Peter; Zorec, Robert; Iglic, Ales

2012-12-01

To investigate morphological alterations of malignant cancer cells (T24) of urothelial origin seeded on flat titanium (Ti) and nanotubular TiO(2) (titanium dioxide) nanostructures. Using anodization method, TiO(2) surfaces composed of vertically aligned nanotubes of 50-100 nm diameters were produced. The flat Ti surface was used as a reference. The alteration in the morphology of cancer cells was evaluated using scanning electron microscopy (SEM). A computational model, based on the theory of membrane elasticity, was constructed to shed light on the biophysical mechanisms responsible for the observed changes in the contact area of adhesion. Large diameter TiO(2) nanotubes exhibited a significantly smaller contact area of adhesion (P<0.0001) and had more membrane protrusions (eg, microvilli and intercellular membrane nanotubes) than on flat Ti surface. Numerical membrane dynamics simulations revealed that the low adhesion energy per unit area would hinder the cell spreading on the large diameter TiO(2) nanotubular surface, thus explaining the small contact area. The reduction in the cell contact area in the case of large diameter TiO(2) nanotube surface, which does not enable formation of the large enough number of the focal adhesion points, prevents spreading of urothelial cells.

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

PubMed

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Bone-Immune Cell Crosstalk: Bone Diseases

PubMed Central

Mori, Giorgio; D'Amelio, Patrizia; Faccio, Roberta

2015-01-01

Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma. PMID:26000310

Bone-immune cell crosstalk: bone diseases.

PubMed

Mori, Giorgio; D'Amelio, Patrizia; Faccio, Roberta; Brunetti, Giacomina

2015-01-01

Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma.

Allogeneic substitution for nominal antigen-specific T-cell clone reactivity in schistosomiasis.

PubMed Central

Linette, G P; Lammie, P J; Phillips, S M

1986-01-01

The present studies have established the nature of a T-cell clone which demonstrates dual reactivity directed against Schistosoma mansoni antigen presented by syngeneic antigen presenting cells and against allogeneic cells. Clone G4, when stimulated by either antigen (SEA) or allogeneic cells (PL/J), exhibits similar functional and phenotypic characteristics. A subclone of G4, G4A.1, which has been maintained in continuous mixed lymphocyte culture for 12 months (in the absence of SEA), retains comparable reactivity with respect to proliferation and ability to produce lymphokines, transfer delayed-type hypersensitivity, and produce in vitro granulomas in response to SEA. Normal antigenic stimulation is highly contingent upon I-Ab compatibility while antibody blocking experiments map allo-reactivity to I-Eu. The failure of B10.PL spleen cells to stimulate G4, however, suggests that alloreactivity may be directed against the recently described Mls X locus. Both allogeneic and nominal antigen induced T-cell activation are blocked by antibody directed against L3T4A, confirming Class II MHC restriction for both types of stimulation. These studies suggest that stimulation of T cells by either alloantigen or nominal antigen elicits qualitatively similar functional profiles, and further suggest the feasibility of producing large numbers of nominal antigen reactive cloned T cells in the absence of nominal antigen under mixed lymphocyte culture conditions. PMID:2420707

Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells.

PubMed

Qiu, Caihong; Olivier, Emmanuel N; Velho, Michelle; Bouhassira, Eric E

2008-02-15

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.

Measurement of Device Parameters Using Image Recovery Techniques in Large-Scale IC Devices

NASA Technical Reports Server (NTRS)

Scheick, Leif; Edmonds, Larry

2004-01-01

Devices that respond to radiation on a cell level will produce histograms showing the relative frequency of cell damage as a function of damage. The measured distribution is the convolution of distributions from radiation responses, measurement noise, and manufacturing parameters. A method of extracting device characteristics and parameters from measured distributions via mathematical and image subtraction techniques is described.

Selective Laser Melting: a regular unit cell approach for the manufacture of porous, titanium, bone in-growth constructs, suitable for orthopedic applications.

PubMed

Mullen, Lewis; Stamp, Robin C; Brooks, Wesley K; Jones, Eric; Sutcliffe, Christopher J

2009-05-01

In this study, a novel porous titanium structure for the purpose of bone in-growth has been designed, manufactured and evaluated. The structure was produced by Selective Laser Melting (SLM); a rapid manufacturing process capable of producing highly intricate, functionally graded parts. The technique described utilizes an approach based on a defined regular unit cell to design and produce structures with a large range of both physical and mechanical properties. These properties can be tailored to suit specific requirements; in particular, functionally graded structures with bone in-growth surfaces exhibiting properties comparable to those of human bone have been manufactured. The structures were manufactured and characterized by unit cell size, strand diameter, porosity, and compression strength. They exhibited a porosity (10-95%) dependant compression strength (0.5-350 Mpa) comparable to the typical naturally occurring range. It is also demonstrated that optimized structures have been produced that possesses ideal qualities for bone in-growth applications and that these structures can be applied in the production of orthopedic devices. (c) 2008 Wiley Periodicals, Inc.

Recombinant protein vaccines produced in insect cells.

PubMed

Cox, Manon M J

2012-02-27

The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises. Copyright © 2012 Elsevier Ltd. All rights reserved.

Advances in polycrystalline thin-film photovoltaics for space applications

NASA Technical Reports Server (NTRS)

Lanning, Bruce R.; Armstrong, Joseph H.; Misra, Mohan S.

1994-01-01

Polycrystalline, thin-film photovoltaics represent one of the few (if not the only) renewable power sources which has the potential to satisfy the demanding technical requirements for future space applications. The demand in space is for deployable, flexible arrays with high power-to-weight ratios and long-term stability (15-20 years). In addition, there is also the demand that these arrays be produced by scalable, low-cost, high yield, processes. An approach to significantly reduce costs and increase reliability is to interconnect individual cells series via monolithic integration. Both CIS and CdTe semiconductor films are optimum absorber materials for thin-film n-p heterojunction solar cells, having band gaps between 0.9-1.5 ev and demonstrated small area efficiencies, with cadmium sulfide window layers, above 16.5 percent. Both CIS and CdTe polycrystalline thin-film cells have been produced on a laboratory scale by a variety of physical and chemical deposition methods, including evaporation, sputtering, and electrodeposition. Translating laboratory processes which yield these high efficiency, small area cells into the design of a manufacturing process capable of producing 1-sq ft modules, however, requires a quantitative understanding of each individual step in the process and its (each step) effect on overall module performance. With a proper quantification and understanding of material transport and reactivity for each individual step, manufacturing process can be designed that is not 'reactor-specific' and can be controlled intelligently with the design parameters of the process. The objective of this paper is to present an overview of the current efforts at MMC to develop large-scale manufacturing processes for both CIS and CdTe thin-film polycrystalline modules. CIS cells/modules are fabricated in a 'substrate configuration' by physical vapor deposition techniques and CdTe cells/modules are fabricated in a 'superstrate configuration' by wet chemical methods. Both laser and mechanical scribing operations are used to monolithically integrate (series interconnect) the individual cells into modules. Results will be presented at the cell and module development levels with a brief description of the test methods used to qualify these devices for space applications. The approach and development efforts are directed towards large-scale manufacturability of established thin-film, polycrystalline processing methods for large area modules with less emphasis on maximizing small area efficiencies.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1990-01-01

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Accelerator-driven boron neutron capture therapy

NASA Astrophysics Data System (ADS)

Edgecock, Rob

2014-05-01

Boron Neutron Capture Therapy is a binary treatment for certain types of cancer. It works by loading the cancerous cells with a boron-10 carrying compound. This isotope has a large cross-section for thermal neutrons, the reaction producing a lithium nucleus and alpha particle that kill the cell in which they are produced. Recent studies of the boron carrier compound indicate that the uptake process works best in particularly aggressive cancers. Most studied is glioblastoma multiforme and a trial using a combination of BNCT and X-ray radiotherapy has shown an increase of nearly a factor of two in mean survival over the state of the art. However, the main technical problem with BNCT remains producing a sufficient flux of neutrons for a reasonable treatment duration in a hospital environment. This paper discusses this issue.

γδ T Cells Shape Pre-Immune Peripheral B Cell Populations

PubMed Central

Huang, Yafei; Getahun, Andrew; Heiser, Ryan A.; Detanico, Thiago O.; Aviszus, Katja; Kirchenbaum, Greg A.; Casper, Tamara L.; Huang, Chunjian; Aydintug, M. Kemal; Carding, Simon R.; Ikuta, Koichi; Huang, Hua; Wysocki, Lawrence J.; Cambier, John C.; O’Brien, Rebecca L.; Born, Willi K.

2015-01-01

We previously reported that selective ablation of certain γδ T cell subsets rather than removal of all γδ T cells, strongly affects serum antibody levels in non-immunized mice. This type of manipulation also changed T cells including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4+ and Vγ6+ γδ T cells (B6.TCR-Vγ4−/−/6−/−), we observed expanded Vγ1+ cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4−/−/6−/− mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of antibody-producing cells, and serum levels of antibodies, IL-4 and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain, and their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Together, these data demonstrate the capability of γδ T cells of modulating size and productivity of pre-immune peripheral B cell populations. PMID:26582947

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

PubMed

Huang, Yafei; Getahun, Andrew; Heiser, Ryan A; Detanico, Thiago O; Aviszus, Katja; Kirchenbaum, Greg A; Casper, Tamara L; Huang, Chunjian; Aydintug, M Kemal; Carding, Simon R; Ikuta, Koichi; Huang, Hua; Wysocki, Lawrence J; Cambier, John C; O'Brien, Rebecca L; Born, Willi K

2016-01-01

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations. Copyright © 2015 by The American Association of Immunologists, Inc.

Single-cell analysis of lymphokine imbalance in asymptomatic HIV-1 infection: evidence for a major alteration within the CD8+ T cell subset

PubMed Central

Sousa, A E; Victorino, R M M

1998-01-01

In this study we investigated at single-cell level by flow cytometry the potential of T cell cytokine production in asymptomatic HIV-1-infected subjects with > 200 CD4 counts and possible correlation with T helper cell depletion and viral load. Mitogen-stimulated peripheral blood mononuclear cells from 32 HIV-1+ patients and 16 healthy subjects were intracytoplasmically stained for IL-2, interferon-gamma (IFN-γ), IL-4 or IL-10, and the frequency of cytokine-producing cells was assessed in total T cells, CD4, CD8 and CD45RO subsets as well as in CD69+CD3+ gated lymphocytes. HIV-1+ patients, irrespective of their degree of CD4 depletion, exhibited a major increase in IFN-γ+ CD8 T cells, largely due to CD28− cells, as well as a decrease in the capacity of CD8 T cells to produce IL-2. Patients with > 500 CD4 counts showed a diminished frequency of IL-4 expression in CD4 T cells and a negative correlation was found between this parameter and the ex vivo CD4 counts in the 32 patients. Analysis of patients stratified according to viral load revealed a significantly higher proportion of IL-2-producing CD4 cells in the group with < 5000 RNA copies/ml. In short, using single-cell analysis and an antigen-presenting cell-independent stimulus, we have not been able to find any significant cytokine imbalances in the CD4 subset, suggesting that the well described T helper defects are not due to intrinsic alterations in the potential of CD4 T cells to produce cytokines. On the other hand, the major disturbances in the CD8 T lymphocytes agree with the marked activation and possible replicative senescence of CD8 T cells and emphasize the role of this subset in HIV immunopathogenesis. PMID:9649194

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

NASA Astrophysics Data System (ADS)

Nandi, Saroj Kumar; Safran, Sam A.

2018-05-01

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

Donor-Specific Antibodies Are Produced Locally in Ectopic Lymphoid Structures in Cardiac Allografts.

PubMed

Huibers, M M H; Gareau, A J; Beerthuijzen, J M T; Siera-de Koning, E; van Kuik, J; Kamburova, E G; Vink, A; de Jonge, N; Lee, T D G; Otten, H G; de Weger, R A

2017-01-01

Cardiac allograft vasculopathy (CAV) is a transplant pathology, limiting graft survival after heart transplantation. CAV arteries are surrounded by ectopic lymphoid structures (ELS) containing B cells and plasma cells. The aim of this study was to characterize the antigenic targets of antibodies produced in ELS. Coronary arteries and surrounding epicardial tissue from 56 transplant recipients were collected during autopsy. Immunofluorescence was used to identify antibody-producing plasma cells. Immunoglobulin levels in tissue lysates were measured by enzyme-linked immunosorbent assay and analyzed for donor-specific HLA antibodies by Luminex assay. Cytokine and receptor expression levels were quantified using quantitative polymerase chain reaction. Plasma cells in ELS were polyclonal and produced IgG and/or IgM antibodies. In epicardial tissue, IgG (p < 0.05) and IgM levels were higher in transplant patients with larger ELS than smaller ELS. In 4 of 21 (19%) patients with ELS, donor-specific HLA type II antibodies were detected locally. Cytokine and receptor expression (CXCR3, interferon γ and TGF-β) was higher in large ELS in the epicardial tissue than in other vessel wall layers, suggesting active recruitment and proliferation of T and B lymphocytes. ELS exhibited active plasma cells producing locally manufactured antibodies that, in some cases, were directed against the donor HLA, potentially mediating rejection with major consequences for the graft. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Production of large numbers of hybridomas producing monoclonal antibodies against rat IgE using mast cell-deficient w/wv and sl/sld strains of mice.

PubMed

Rup, B J

1989-08-15

A number of different mouse strains and immunization protocols were used to attempt to make monoclonal antibodies against rat IgE for use in studies of the structure, biological activities and regulation of this class of antibody. Successful production of large numbers of monoclonal antibodies was achieved when mast cell deficient (w/wv and sl/sld) but not conventional (BALB/c, CAF1 or SJL) mice were used. These results suggest that the poor response of conventional strains of mice to rat IgE may be due to the presence of mast cells bearing high affinity receptors for IgE in these mice.

Microfluidic approach of Sickled Cell Anemia

NASA Astrophysics Data System (ADS)

Abkarian, Manouk; Loiseau, Etienne; Massiera, Gladys

2012-11-01

Sickle Cell Anemia is a disorder of the microcirculation caused by a genetic point mutation that produces an altered hemoglobin protein called HbS. HbS self-assembles reversibly into long rope like fibers inside the red blood cells. The resulting distorded sickled red blood cells are believed to block the smallest capillaries of the tissues producing anemia. Despite the large amount of work that provided a thorough understanding of HbS polymerization in bulk as well as in intact red blood cells at rest, no consequent cellular scale approaches of the study of polymerization and its link to the capillary obstruction have been proposed in microflow, although the problem of obstruction is in essence a circulatory problem. Here, we use microfluidic channels, designed to mimic physiological conditions (flow velocity, oxygen concentration, hematocrit...) of the microcirculation to carry out a biomimetic study at the cellular scale of sickled cell vaso-occlusion. We show that flow geometry, oxygen concentration, white blood cells and free hemoglobin S are essential in the formation of original cell aggregates which could play a role in the vaso-occlusion events.

The remarkable occurrence of large rainfall-induced debris flows at two different locations on July 12, 2008, Southern Sierra Nevada, CA, USA

USGS Publications Warehouse

DeGraff, J.V.; Wagner, D.L.; Gallegos, A.J.; DeRose, M.; Shannon, C.; Ellsworth, T.

2011-01-01

On July 12, 2008, two convective cells about 155 km apart produced a brief period of intense rainfall triggering large debris flows in the southern Sierra Nevada. The northernmost cell was centered over Oak Creek Canyon, an east-flowing drainage, and its tributaries near Independence, CA, USA. About 5:00 P.M., debris flows passed down the South Fork and North Fork of Oak Creek to merge into a large single feature whose passage affected the historic Mt. Whitney Fish hatchery and blocked California State Highway 395. At about the same time, the southernmost cell was largely centered over Erskine Creek, a main tributary of the west-flowing Kern River. Debris flows issued from several branches to coalesce into a large debris flow that passed along Erskine Creek, through the town of Lake Isabella, CA, USA and into the Kern River. It was observed reaching Lake Isabella about 6:30 P.M. Both debris flows caused significant disruption and damage to local communities. ?? 2011 Springer-Verlag.

Evaluation of Production Version of the NASA Improved Inorganic-Organic Separator

NASA Technical Reports Server (NTRS)

Sheibley, D.

1983-01-01

The technology of an inorganic-organic (I/O) separator, which demonstrated improved flexibility, reduced cost, production feasibility and improved cycle life was developed. Substrates to replace asbestos and waterbased separator coatings to replace the solvent based coatings were investigated. An improved fuel cell grade asbestos sheet was developed and a large scale production capability for the solvent based I/O separator was demonstrated. A cellulose based substrate and a nonwoven polypropylene fiber substrate were evaluated as replacements for the asbestos. Both the cellulose and polypropylene substrates were coated with solvent based and water based coatings to produce a modified I/O separator. The solvent based coatings were modified to produce aqueous separator coatings with acceptable separator properties. A single ply fuel cell grade asbestos with a binder (BTA) was produced. It has shown to be an acceptable substrate for the solvent and water based separator coatings, an acceptable absorber for alkaline cells, and an acceptable matrix for alkaline fuel cells. The original solvent based separator (K19W1), using asbestos as a substrate, was prepared.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Quantitative Assessment of Neurite Outgrowth in PC12 Cells

EPA Science Inventory

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes such as neuronal differenti...

Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

PubMed

Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

2014-01-01

Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

Production technology for high efficiency ion implanted solar cells

NASA Technical Reports Server (NTRS)

Kirkpatrick, A. R.; Minnucci, J. A.; Greenwald, A. C.; Josephs, R. H.

1978-01-01

Ion implantation is being developed for high volume automated production of silicon solar cells. An implanter designed for solar cell processing and able to properly implant up to 300 4-inch wafers per hour is now operational. A machine to implant 180 sq m/hr of solar cell material has been designed. Implanted silicon solar cells with efficiencies exceeding 16% AM1 are now being produced and higher efficiencies are expected. Ion implantation and transient processing by pulsed electron beams are being integrated with electrostatic bonding to accomplish a simple method for large scale, low cost production of high efficiency solar cell arrays.

Chemical vapor deposition growth

NASA Technical Reports Server (NTRS)

Ruth, R. P.; Manasevit, H. M.; Kenty, J. L.; Moudy, L. A.; Simpson, W. I.; Yang, J. J.

1976-01-01

The chemical vapor deposition (CVD) method for the growth of Si sheet on inexpensive substrate materials is investigated. The objective is to develop CVD techniques for producing large areas of Si sheet on inexpensive substrate materials, with sheet properties suitable for fabricating solar cells meeting the technical goals of the Low Cost Silicon Solar Array Project. Specific areas covered include: (1) modification and test of existing CVD reactor system; (2) identification and/or development of suitable inexpensive substrate materials; (3) experimental investigation of CVD process parameters using various candidate substrate materials; (4) preparation of Si sheet samples for various special studies, including solar cell fabrication; (5) evaluation of the properties of the Si sheet material produced by the CVD process; and (6) fabrication and evaluation of experimental solar cell structures, using standard and near-standard processing techniques.

Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

PubMed

Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

2009-10-30

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

Comparison Through Image Analysis Between Al Foams Produced Using Two Different Methods

NASA Astrophysics Data System (ADS)

Boschetto, A.; Campana, F.; Pilone, D.

2014-02-01

Several methods are available for making metal foams. They allow to tailor their mechanical, thermal, acoustic, and electrical properties for specific applications by varying the relative density as well as the cell size and morphology. Foams have a very heterogeneous structure so that their properties may show a large scatter. In this paper, an aluminum foam produced by means of foaming of powder compacts and another one prepared via the infiltration process were analyzed and compared. Image analysis has been used as a useful tool to determine size, morphology, and distribution of cells in both foams and to correlate cell morphology with the considered manufacturing process. The results highlighted that cell size and morphology are strictly dependent upon the manufacturing method. This paper shows how some standard 2D morphological indicators may be usefully adopted to characterize foams whose structure derives from the specific manufacturing process.

Spectrum of mucin-producing neoplastic conditions of the abdomen and pelvis: Cross-sectional imaging evaluation

PubMed Central

Lee, Nam Kyung; Kim, Suk; Kim, Hyun Sung; Jeon, Tae Yong; Kim, Gwang Ha; Kim, Dong Uk; Park, Do Youn; Kim, Tae Un; Kang, Dae Hwan

2011-01-01

Various mucin-producing neoplasms originate in different abdominal and pelvic organs. Mucinous neoplasms differ from non-mucinous neoplasms because of the differences in clinical outcome and imaging appearance. Mucinous carcinoma, in which at least 50% of the tumor is composed of large pools of extracellular mucin and columns of malignant cells, is associated with a worse prognosis. Signet ring cell carcinoma is characterized by large intracytoplasmic mucin vacuoles that expand in the malignant cells with the nucleus displaced to the periphery. Its prognosis is also generally poor. In contrast, intraductal papillary mucinous neoplasm of the bile duct and pancreas, which is characterized by proliferation of ductal epithelium and variable mucin production, has a better prognosis than other malignancies in the pancreaticobiliary tree. Imaging modalities play a critical role in differentiating mucinous from non-mucinous neoplasms. Due to high water content, mucin has a similar appearance to water on ultrasound (US), computed tomography (CT), and magnetic resonance imaging, except when thick and proteinaceous, and then it tends to be hypoechoic with fine internal echoes or have complex echogenicity on US, hyperdense on CT, and hyperintense on T1- and hypointense on T2-weighted images, compared to water. Therefore, knowledge of characteristic mucin imaging features is helpful to diagnose various mucin-producing neoplastic conditions and to facilitate appropriate treatment. PMID:22147976

Whodunit? The Contribution of Interleukin (IL)-17/IL-22-Producing γδ T Cells, αβ T Cells, and Innate Lymphoid Cells to the Pathogenesis of Spondyloarthritis

PubMed Central

Reinhardt, Annika; Prinz, Immo

2018-01-01

γδ T cells, αβ T cells, and innate lymphoid cells (ILCs) are capable of producing interleukin (IL)-17A, IL-17F, and IL-22. Among these three families of lymphocytes, it is emerging that γδ T cells are, at least in rodents, the main source of these key pro-inflammatory cytokines. γδ T cells were implicated in multiple inflammatory and autoimmune diseases, including psoriasis, experimental autoimmune encephalomyelitis and uveitis, colitis, and rheumatoid arthritis. Recent findings pointed toward a central role of γδ T cells in the pathogenesis of spondyloarthritis (SpA), a group of inflammatory rheumatic diseases affecting the axial skeleton. SpA primarily manifests as inflammation and new bone formation at the entheses, which are connecting tendons or ligaments with bone. In SpA patients, joint inflammation is frequently accompanied by extra-articular manifestations, such as inflammatory bowel disease or psoriasis. In humans, genome-wide association studies could link the IL-23/IL-17 cytokine axis to SpA. Accordingly, antibodies targeting IL-23/IL-17 for SpA treatment already showed promising results in clinical studies. However, the contribution of IL-17-producing γδ T cells to SpA pathogenesis is certainly not an open-and-shut case. Indeed, the cell types that are chiefly involved in local inflammation in human SpA still remain largely unclear. Some studies focusing on blood or synovium from SpA patients reported augmented IL-17-producing and IL-23 receptor-expressing γδ T cells, but other cell types might contribute as well. Here, we summarize the current understanding of how γδ T cells, αβ T cells, and ILCs contribute to the pathogenesis of human and experimental SpA. PMID:29922283

An unusual variant of Trichophyton tonsurans var. sulfureum.

PubMed

Padhye, A A; Weitzman, I; Domenech, E

1994-01-01

A fungus, recovered from a skin lesion of a patient, produced velvety to powdery, white to deep yellow colonies on Sabouraud glucose agar. Microscopically, it produced a large number of cylindric, smooth-walled, three- to eight-celled macroconidia but failed to produce microconidia on a variety of nutritional media such as rice grains, cornmeal dextrose, potato dextrose, Sabouraud glucose, oatmeal and lactrimel agars. It hydrolysed urea in 7 days, perforated hair in vitro and required thiamine for growth. This isolate represents an atypical variant of Trichophyton tonsurans var. sufureum subvar. perforans.

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

PubMed

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

2017-11-24

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

Recent advances in understanding of meiosis initiation and the apomictic pathway in plants.

PubMed

Wang, Chung-Ju R; Tseng, Ching-Chih

2014-01-01

Meiosis, a specialized cell division to produce haploid cells, marks the transition from a sporophytic to a gametophytic generation in the life cycle of plants. In angiosperms, meiosis takes place in sporogenous cells that develop de novo from somatic cells in anthers or ovules. A successful transition from the mitotic cycle to the meiotic program in sporogenous cells is crucial for sexual reproduction. By contrast, when meiosis is bypassed or a mitosis-like division occurs to produce unreduced cells, followed by the development of an embryo sac, clonal seeds can be produced by apomixis, an asexual reproduction pathway found in 400 species of flowering plants. An understanding of the regulation of entry into meiosis and molecular mechanisms of apomictic pathway will provide vital insight into reproduction for plant breeding. Recent findings suggest that AM1/SWI1 may be the key gene for entry into meiosis, and increasing evidence has shown that the apomictic pathway is epigenetically controlled. However, the mechanism for the initiation of meiosis during sexual reproduction or for its omission in the apomictic pathway still remains largely unknown. Here we review the current understanding of meiosis initiation and the apomictic pathway and raised several questions that are awaiting further investigation.

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

PubMed Central

Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

2013-01-01

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. PMID:24312821

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

PubMed

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.

Highly Tumorigenic Diffuse Large B Cell Lymphoma Cells Are Produced by Coculture with Stromal Cells.

PubMed

Lin, Zhiguang; Chen, Bobin; Wu, Ting; Xu, Xiaoping

2018-05-23

Diffuse large B cell lymphoma (DLBCL) is heterogeneous. We aimed to explore how tumor microenvironment promotes lymphoma cell aggressiveness and heterogeneity. We created a coculture system using human DLBCL cells and mouse bone marrow stromal cells. Proliferative capacity, drug resistance, clonogenicity, and tumorigenicity were compared in lymphoma cells from the coculture system and lymphoma cells cultured alone. Expression of Notch signaling associated genes was evaluated using real-time reverse transcriptase PCR and Western blot. Lymphoma cells in the coculture system differentiated into a suspended cell group and an adherent cell group. They acquired a stronger proliferative capacity and drug resistance than lymphoma cells cultured alone, and differences existed between the adherent cell and suspended cell groups. The suspended cell group acquired the most powerful clonogenic and tumorigenic potential. However, Notch3 was exclusively expressed in the adherent lymphoma cell group and the use of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, an inhibitor of Notch pathway, could abolish the emergence of highly aggressive lymphoma cells. Highly tumorigenic lymphoma cells could be generated by coculture with stromal cells, and it was dependent on Notch3 expression in the adjacent lymphoma cells through interaction with stromal cells. © 2018 S. Karger AG, Basel.

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

PubMed

Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

2013-08-01

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Recent advances in our understanding of giant cell arteritis pathogenesis.

PubMed

Samson, Maxime; Corbera-Bellalta, Marc; Audia, Sylvain; Planas-Rigol, Ester; Martin, Laurent; Cid, Maria Cinta; Bonnotte, Bernard

2017-08-01

Giant cell arteritis (GCA) is a granulomatous vasculitis affecting large arteries, especially the aorta and the extracranial branches of the external carotid artery. Its exact pathogenesis is not fully understood but major progress has been made in recent years, leading to new therapeutic targets like inhibition of the interleukin-6 pathway or the modulation of immune checkpoints. The cause of GCA has not been clearly identified but it is thought that GCA occurs on a genetic background and is triggered by unknown environmental factors that could activate and lead to the maturation of dendritic cells localized in the adventitia of normal arteries. These activated dendritic cells then produce chemokines which trigger the recruitment of CD4 + T cells, which in turn become activated, proliferate and polarize into Th1 and Th17 cells, which produce IFN-γ and IL-17, respectively. Exposed to IFN-γ, endothelial cells and vascular smooth muscle cells produce chemokines leading to the recruitment of further Th1 cells, CD8 + T cells and monocytes. The latter differentiate into macrophages, which, when persistently exposed to IFN-γ, form giant cells, the histological hallmark of GCA. With the contribution of vascular smooth muscle cells, immune cells then trigger the destruction and remodeling of the arterial wall, thus leading to the formation of a neo-intima resulting in progressive occlusion of the arterial lumen, which is responsible for the ischemic symptoms of GCA. In this paper, we review recent progress in our understanding of GCA pathogenesis in the fields of genetics, epigenetics, infections, immunology and vascular remodeling. Copyright © 2017 Elsevier B.V. All rights reserved.

Ion Implantation with in-situ Patterning for IBC Solar Cells

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

Graff, John W.

2014-10-24

Interdigitated back-side Contact (IBC) solar cells are the highest efficiency silicon solar cells currently on the market. Unfortunately the cost to produce these solar cells is also very high, due to the large number of processing steps required. Varian believes that only the combination of high efficiency and low cost can meet the stated goal of $1/Wp. The core of this program has been to develop an in-situ patterning capability for an ion implantation system capable of producing patterned doped regions for IBC solar cells. Such a patterning capable ion implanter can reduce the number of process steps required tomore » manufacture IBC cells, and therefore significantly reduce the cost. The present program was organized into three phases. Phase I was to select a patterning approach and determine the patterning requirements for IBC cells. Phase II consists of construction of a Beta ion implantation system containing in-situ patterning capability. Phase III consists of shipping and installation of the ion implant system in a customer factory where it will be tested and proven in a pilot production line.« less

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

PubMed

Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

2016-01-01

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

PubMed

Cervera, Laura; Kamen, Amine A

2018-01-01

Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1 × 10 6 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.

Limitations of a hemolytic plaque assay for IgG-anti-IgG rheumatoid factor-producing cells.

PubMed

Venn, A J; Dresser, D W

1987-09-24

An attempt has been made to develop a hemolytic plaque assay capable of detecting homophile IgG rheumatoid factor (RF)-producing cells. Anti-immunoglobulin allotype-developing reagents were used to distinguish between target and effector IgG. The hemolytic assay has been used to demonstrate an apparently high level of homophile IgM and IgG RF-producing cells in the spleens and lymph nodes of mice stimulated by LPS. However, it appears that a large proportion of the plaques obtained in these assays are due to an artefact resulting from cross-linking of target and effector molecules by the developing reagents. In the case of IgM RF the artefact depends on the presence of a small contamination of the target IgG by IgM, allowing cross-linking of target and effector IgM by the anti-mu-specific developing reagent. With the IgG RF, cross-reactivity of the rabbit anti-Ighb allotype-developing serum for the 'wrong' (Igha) allotype, normally undetectable, becomes sufficient to be biologically relevant when the developing antibody is complexed by being bound to its target (Ighb) allotype. Nevertheless anti-allotype reagents may afford an accurate means of detecting homophile IgG RF producing cells using other assay systems.

Development of the Statocyst in Aplysia Californica. Part 1; Observations on Statoconial Development

NASA Technical Reports Server (NTRS)

Wiederhold, Michael L.; Sharma, Jyotsna S.; Driscoll, Brian P.; Harrison, Jeffrey L.

1990-01-01

The gravity receptor organs of gastropod molluscs, such as Aplysia californica, are bilateral paired statocysts, which contain dense statoconia within a fluid-filled cyst. Gravitational forces on the statoconia are sensed through their interaction with ciliated mechanoreceptor cells in the wall of the cyst. Larval Aplysia contain a single statolith within each statocyst; when the animals grow to a critical size, they begin producing multiple statoconia, a process that continues throughout life. The number of statoconia is highly correlated with animal weight but poorly correlated with age, indicating that stone production is related to total metabolism. The single statolith has an amorphous internal structure whereas the multiple statoconia have calcification deposited on concentric layers of membrane or matrix protein. The statolith appears to be produced within the cyst lumen but the multiple statoconia are produced within supporting cells between the receptor cells. Large adult animals have statoconia larger than those in early post-metamorphic animals which have just started producing multiple stones. The maximum statocyst diameter at which the receptor-cell cilia can suspend the statolith in the center of the cyst lumen is 45 micrometers; production of multiple stones begins when the cyst reaches this size. The mechanisms by which statoconia production is initiated and controlled are discussed.

Enteroaggregative, Shiga Toxin-Producing Escherichia coli O111:H2 Associated with an Outbreak of Hemolytic-Uremic Syndrome

PubMed Central

Morabito, Stefano; Karch, Helge; Mariani-Kurkdjian, Patrizia; Schmidt, Herbert; Minelli, Fabio; Bingen, Edouard; Caprioli, Alfredo

1998-01-01

Shiga toxin-producing Escherichia coli O111:H2 strains from an outbreak of hemolytic-uremic syndrome showed aggregative adhesion to HEp-2 cells and harbored large plasmids which hybridized with the enteroaggregative E. coli probe PCVD432. These strains present a novel combination of virulence factors and might be as pathogenic to humans as the classic enterohemorrhagic E. coli. PMID:9508328

Silicon-on-ceramic process: Silicon sheet growth and device development for the large-area silicon sheet task of the low-cost solar array project

NASA Technical Reports Server (NTRS)

Whitehead, A. B.; Zook, J. D.; Grung, B. L.; Heaps, J. D.; Schmit, F.; Schuldt, S. B.; Chapman, P. W.

1981-01-01

The technical feasibility of producing solar cell quality sheet silicon to meet the DOE 1986 cost goal of 70 cents/watt was investigated. The silicon on ceramic approach is to coat a low cost ceramic substrate with large grain polycrystalline silicon by unidirectional solidification of molten silicon. Results and accomplishments are summarized.

Involvement of granulysin-producing T cells in the development of superficial microbial folliculitis.

PubMed

Oono, T; Morizane, S; Yamasaki, O; Shirafuji, Y; Huh, W-K; Akiyama, H; Iwatsuki, K

2004-05-01

Granulysin is a recently identified antimicrobial protein expressed on cytotoxic T cells, natural killer (NK) cells and NKT cells. It has been shown that granulysin contributes to the defence mechanisms against mycobacterial infection. Superficial microbial folliculitis is a common skin disease. In a previous report, we showed that, as a first line of defence, alpha-defensin, a human neutrophil peptide, and beta-defensin (human beta-defensin-2) were expressed in infiltrating neutrophils and in lesional epidermal keratinocytes, respectively, in superficial folliculitis. As we also observed many infiltrating lymphocytes in lesional dermis, we hypothesized that infiltrating lymphocytes may possess antimicrobial substances, such as granulysin, and play a role in the defence mechanism as a second line of defence. Seven specimens of superficial microbial folliculitis diagnosed clinically and histologically were examined by means of immunohistochemistry. To identify the phenotype of cells expressing granulysin, confocal laser microscopic examination was performed. A dense lymphoid cell infiltrate was observed in pustules, in the perivascular regions. A large number of these lymphoid cells were positive for granulysin. Phenotypically, cells consisted of CD3+ T cells, CD8+ T cells and UCHL-1+ T cells. CD20+ cells and CD56+ cells were not observed. Microscopic examination with a confocal laser showed that the lymphocytes producing granulysin were CD3+ and CD4+ T cells but not CD8+ T cells. We showed that many granulysin-bearing T cells infiltrated affected follicles and perilesional dermis in superficial microbial folliculitis. However, few granulysin-positive lymphoid cells were observed in sterile pustular lesions. Our observations indicated that adaptive immunity such as granulysin, a lymphocyte-produced antimicrobial protein, may play an important role in the cutaneous defence mechanism.

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

PubMed Central

2011-01-01

Background Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system. Results A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml-1 cell cultures) which can oxygenate linoleic acid resulting in high yields (18 μmol ml-1 cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 μmol mg-1 crude protein). The yield of C9-aldehydes from linoleic acid was 64%. Conclusion The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E),12(Z)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent. PMID:21450085

Bioelectrochemical reduction of volatile fatty acids in anaerobic digestion effluent for the production of biofuels.

PubMed

Kondaveeti, Sanath; Min, Booki

2015-12-15

This study proves for the first time the feasibility of biofuel production from anaerobic digestion effluent via bioelectrochemical cell operation at various applied cell voltages (1.0, 1.5 and 2.0 V). An increase in cell voltage from 1 to 2 V resulted in more reduction current generation (-0.48 to -0.78 mA) at a lowered cathode potential (-0.45 to -0.84 mV vs Ag/AgCl). Various alcohols were produced depending on applied cell voltages, and the main products were butanol, ethanol, and propanol. Hydrogen and methane production were also observed in the headspace of the cell. A large amount of lactic acid was unexpectedly formed at all conditions, which might be the primary cause of the limited biofuel production. The addition of neutral red (NR) to the system could increase the cathodic reduction current, and thus more biofuels were produced with an enhanced alcohol formation compared to without a mediator. Copyright © 2015 Elsevier Ltd. All rights reserved.

Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens.

PubMed Central

Gurney, E G; Harrison, R O; Fenno, J

1980-01-01

We have isolated three clones of hybrid cells which synthesize antibodies specific for determinants on simian virus 40 (SV40) T antigens. Mouse myeloma NS1 cells were fused with spleen cells from mice that had been immunized with SV40-transformed mouse cells. Hybrid cells were selected in HAT medium and cloned in soft agar. We used an enzyme-linked immunosorbent assay for detection and quantification of mouse antibodies against SV40 T antigens. Monoclonal antibodies from 3 of the 24 clones that scored as positive in the enzyme-linked immunosorbent assay were verified by immunoprecipitation to be specific for SV40 T antigens. Two clones (7 and 412) produced antibodies that recognized denaturation-sensitive antigenic determinants unique to large T antigen. Antibodies from clone 7 appeared to have a low affinity for large T antigen. Antibodies from clone 412 had a higher affinity for large T antigen but did not recognize a subclass of large T antigen that was recognized by tumor serum. Antibodies of the third clone, clone 122, recognized a denaturation-stable antigenic determinant of the 53,000-dalton mouse nonviral T antigen in SV40-transformed cells. Antibodies from clone 122 also recognized similar (51,000- to 56,000-dalton) nonviral T antigens in SV40-transormed or lytically infected cells from five mammalian species and in four uninfected mouse lines. From these observations, we have concluded that (i) the 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and (ii) the nonviral T antigens of five mammalian species share at least one antigenic determinant. Images PMID:6155477

Pasteurella multocida Toxin Manipulates T Cell Differentiation

PubMed Central

Hildebrand, Dagmar; Heeg, Klaus; Kubatzky, Katharina F.

2015-01-01

Pasteurella multocida causes various diseases in a broad range of wild and domestic animals. Toxigenic strains of the serotypes A and D produce an AB protein toxin named Pasteurella multocida toxin (PMT). PMT constitutively activates the heterotrimeric G protein subunits Gαq, Gα13, and Gαi through deamidation of a glutamine residue, which results in cytoskeletal rearrangements as well as increased proliferation and survival of the host cell. In human monocytes, PMT alters the lipopolysaccharide (LPS)-induced activation toward a phenotype that suppresses T cell activation. Here we describe that the toxin also modulates CD4-positive T helper (Th) cells directly. PMT amplifies the expansion of Th cells through enhanced cell cycle progression and suppression of apoptosis and manipulates the differentiation of Th subclasses through activation of Signal Transducers and Activators of Transcription (STAT) family members and induction of subtype-specific master transcription factors. A large population of toxin-treated T cells is double-positive for Foxp3 and RORγt, the transcription factors expressed by Treg and Th17 cells, respectively. This suggests that these cells could have the potential to turn into Th17 cells or suppressive Treg cells. However, in terms of function, the PMT-differentiated cells behave as inflammatory Th17 cells that produce IL-17 and trigger T cell proliferation. PMID:26635744

Comparative Transcriptomics to Identify Novel Genes and Pathways in Dinoflagellates

NASA Astrophysics Data System (ADS)

Ryan, D.

2016-02-01

The unarmored dinoflagellate Karenia brevis is among the most prominent harmful, bloom-forming phytoplankton species in the Gulf of Mexico. During blooms, the polyketides PbTx-1 and PbTx-2 (brevetoxins) are produced by K. brevis. Brevetoxins negatively impact human health and the Gulf shellfish harvest. However, the genes underlying brevetoxin synthesis are currently unknown. Because the K. brevis genome is extremely large ( 1 × 1011 base pairs long), and with a high proportion of repetitive, non-coding DNA, it has not been sequenced. In fact, large, repetitive genomes are common among the dinoflagellate group. High-throughput RNA sequencing technology enabled us to assemble Karenia transcriptomes de novo and investigate potential genes in the brevetoxin pathway through comparative transcriptomics. The brevetoxin profile varies among K. brevis clonal cultures. For example, well-documented Wilson-CCFWC268 typically produces 8-10 pg PbTx per cell, whereas SP1 produces < 2 pg PbTx/cell, and the mutant low-toxin Wilson clone produces undetectable to low (<0.05 pg/cell) amounts. Further, PbTx-2 has been measured in Karenia papilionacea but not Karenia mikimotoi. We compared the transcriptomes of four K. brevis clones (Wilson-CCFWC268, SP3, SP1, and mutant low-toxin Wilson) with K. papilionacea and K. mikimotoi to investigate nucleotide-level genetic variations and differences in gene expression. Of the 85,000 transcripts in the K. brevis transcriptome, 4,600 transcripts, including novel unannotated orthologs and putative polyketide synthases (PKSs), were only expressed by brevetoxin-producing K. brevis and K. papilionacea, not K. mikimotoi. Examination of gene expression between the typical- and low-toxin Wilson clones identified about 3,500 genes with significantly different expression levels, including 2 putative PKSs. One of the 2 PKSs was only found in the brevetoxin-producing Karenia species. These transcriptomes could not have been characterized without high-throughput RNA sequencing.

Strain Prioritization and Genome Mining for Enediyne Natural Products.

PubMed

Yan, Xiaohui; Ge, Huiming; Huang, Tingting; Hindra; Yang, Dong; Teng, Qihui; Crnovčić, Ivana; Li, Xiuling; Rudolf, Jeffrey D; Lohman, Jeremy R; Gansemans, Yannick; Zhu, Xiangcheng; Huang, Yong; Zhao, Li-Xing; Jiang, Yi; Van Nieuwerburgh, Filip; Rader, Christoph; Duan, Yanwen; Shen, Ben

2016-12-20

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family. Copyright © 2016 Yan et al.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

Regenerative medicine for diabetes: differentiation of human pluripotent stem cells into functional β-cells in vitro and their proposed journey to clinical translation.

PubMed

Bose, Bipasha; Katikireddy, Kishore Reddy; Shenoy, P Sudheer

2014-01-01

Diabetes is a group of metabolic diseases, rising globally at an alarming rate. Type 1 (juvenile diabetes) is the autoimmune version of diabetes where the pancreas is unable to produce insulin, whereas type 2 (adult onset diabetes) is caused due to insulin resistance of the cells. In either of the cases, elevated blood glucose levels are observed which leads to progressive comorbidity like renal failure, cardiovascular disease, retinopathy, etc. Metformin, sulphonyl urea group of drugs, as well as insulin injections are the available therapies. In advanced cases of diabetes, the drug alone or drug in combination with insulin injections are not able to maintain a steady level of blood glucose. Moreover, frequent insulin injections are rather cumbersome for the patient. So, regenerative medicine could be a permanent solution for fighting diabetes. Islet transplantation has been tried with a limited amount of success on a large population of diabetics because of the shortage of cadaveric pancreas. Therefore, the best proposed alternative is regenerative medicine involving human pluripotent stem cell (hPSC)-derived beta islet transplantation which can be obtained in large quantities. Efficient protocols for in vitro differentiation of hPSC into a large number of sustained insulin-producing beta cells for transplantation will be considered to be a giant leap to address global rise in diabetic cases. Although most of the protocols mimic in vivo pancreatic development in humans, considerable amount of lacuna persists for near-perfect differentiation strategies. Moreover, beta islets differentiated from hPSC have not yet been successfully translated under clinical scenario. © 2014 Elsevier Inc. All rights reserved.

IL-2 Produced by CD8+ Immune T cells Can Augment Their IFN-γ Production Independently from Their Proliferation in the Secondary Response to an Intracellular Pathogen

PubMed Central

Sa, Qila; Woodward, Jerold; Suzuki, Yasuhiro

2013-01-01

Chronic infection with Toxoplasma gondii induces a potent resistance against re-infection, and IFN-γ production by CD8+ T cells is crucial for the protective immunity. However, the molecular mechanisms that regulate the secondary response remain to be elucidated. In the present study, we examined the role of IL-2 in IFN-γ production by CD8+ immune T cells in their secondary responses using T. gondii-specific CD8+ T cell hybridomas and splenic CD8+ immune T cells from chronically infected mice. The majority (92 %) of CD8+ T cell hybridomas produced large amounts of IFN-γ only when a low amount (0.5 ng/ml) of exogenous IL-2 was provided in combination with T. gondii antigens. Inhibition of cell proliferation by mitomycin C (MMC) did not affect the enhancing effect of IL-2 on the IFN-γ production, and significant increases in transcription factor T-bet expression were associated with the IL-2-mediated IFN-γ amplification. Splenic CD8+ immune T cells produced similar low levels of IL-2 in the secondary response to T. gondii, and a blocking of IL-2 signaling by anti-IL-2Rα antibody or inhibitors of JAK1 and JAK3 significantly reduced IFN-γ production of the T cells. This IL-2-mediated upregulation of IFN-γ production was observed in MMC-treated CD8+ immune T cells, thus independent from their cell division. Therefore, endogenous IL-2 produced by CD8+ immune T cells can play an important autocrine enhancing role on their IFN-γ production in the secondary responses to T. gondii, suggesting an importance of induction of CD8+ immune T cells with an appropriate IL-2 production for vaccine development. PMID:23359502

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

PubMed

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi

2015-10-13

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells

PubMed Central

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y.; Tanaka, Minoru; Miyajima, Atsushi

2015-01-01

Summary To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM+ cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM+ cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM+ cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. PMID:26365514

Patterns of B-lymphocyte gene expression elicited by lipopolysaccharide mitogen.

PubMed Central

Janossy, G; Snajdr, J; Simak-Ellis, M

1976-01-01

When large proportions of B lymphocytes from the murine spleen are stimulated in vitro by bacterial lipopolysaccharide (LPS) B lymphoblasts with small amounts of intracellular immunoglobulin (Ig) and plasmablasts with large amounts of intracellular Ig concomitantly proliferate. It is likely that B lymphocytes are heterogeneous and LPS activates B cells to express their predetermined functional capacity since bromodeoxyuridine does not inhibit the initiation of Ig synthesis in plasmablasts, and Ig synthesis starts before these cells complete their first mitosis. The results suggest that LPS is a potent polyclonal activator (of a B-cell subset) but it is not a differentiation factor in the sense that it is unable to determine whether its target cell develops extensive endoplasmic reticulum or follows a different pathway. The results do not exclude that modulation of B cells' genetic programming might take place during T cell-dependent B-lymphocyte activation. The observed B-cell heterogeneity offers a possible explanation for the concomitant emergence of B memory cells and antibody producers during the early phase of immune responses in vivo. Images Figure 3 Figure 5 Figure 7 Figure 8 PMID:1088414

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

PubMed

Brueggeman, Andrew J; Kuehler, Daniel; Weeks, Donald P

2014-09-01

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Magnetic characterization of isolated candidate vertebrate magnetoreceptor cells

PubMed Central

Eder, Stephan H.K.; Cadiou, Hervé; Muhamad, Airina; McNaughton, Peter A.; Kirschvink, Joseph L.; Winklhofer, Michael

2012-01-01

Over the past 50 y, behavioral experiments have produced a large body of evidence for the existence of a magnetic sense in a wide range of animals. However, the underlying sensory physiology remains poorly understood due to the elusiveness of the magnetosensory structures. Here we present an effective method for isolating and characterizing potential magnetite-based magnetoreceptor cells. In essence, a rotating magnetic field is employed to visually identify, within a dissociated tissue preparation, cells that contain magnetic material by their rotational behavior. As a tissue of choice, we selected trout olfactory epithelium that has been previously suggested to host candidate magnetoreceptor cells. We were able to reproducibly detect magnetic cells and to determine their magnetic dipole moment. The obtained values (4 to 100 fAm2) greatly exceed previous estimates (0.5 fAm2). The magnetism of the cells is due to a μm-sized intracellular structure of iron-rich crystals, most likely single-domain magnetite. In confocal reflectance imaging, these produce bright reflective spots close to the cell membrane. The magnetic inclusions are found to be firmly coupled to the cell membrane, enabling a direct transduction of mechanical stress produced by magnetic torque acting on the cellular dipole in situ. Our results show that the magnetically identified cells clearly meet the physical requirements for a magnetoreceptor capable of rapidly detecting small changes in the external magnetic field. This would also explain interference of ac powerline magnetic fields with magnetoreception, as reported in cattle. PMID:22778440

Whole Blood Activation Results in Enhanced Detection of T Cell and Monocyte Cytokine Production by Flow Cytometry

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Crucian, Brian E.

2001-01-01

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a wholeblood activation culture has been described. We compared whole blood culture to standard PBMC culture and determined the individual cytokine secretion patterns for both T cells and monocytes via flow cytometry. For T cells cytokine assessment following PMA +ionomycin activation: (1) a significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture; (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. In addition, a four-color cytometric analysis was used to allow accurate phenotyping and quantitation of cytokine producing lymphocyte populations. Using this technique we found IFNgamma production to be significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines in conjunction with CD 14. The cytokine pairs used for analysis were IL-1a/IL-12, and IL-10ITNFa. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFalpha equally well in both culture systems. Monocyte production of IL-10 was significantly elevated following whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent functionally different monocyte subsets with distinct functions. Whole blood culture eliminates the need to purify cell populations prior to culture and may have significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. It is likely that the altered cytokine production observed following whole blood culture more accurately represents the in-vivo immune balance.

Mesoderm Lineage 3D Tissue Constructs Are Produced at Large-Scale in a 3D Stem Cell Bioprocess.

PubMed

Cha, Jae Min; Mantalaris, Athanasios; Jung, Sunyoung; Ji, Yurim; Bang, Oh Young; Bae, Hojae

2017-09-01

Various studies have presented different approaches to direct pluripotent stem cell differentiation such as applying defined sets of exogenous biochemical signals and genetic/epigenetic modifications. Although differentiation to target lineages can be successfully regulated, such conventional methods are often complicated, laborious, and not cost-effective to be employed to the large-scale production of 3D stem cell-based tissue constructs. A 3D-culture platform that could realize the large-scale production of mesoderm lineage tissue constructs from embryonic stem cells (ESCs) is developed. ESCs are cultured using our previously established 3D-bioprocess platform which is amenable to mass-production of 3D ESC-based tissue constructs. Hepatocarcinoma cell line conditioned medium is introduced to the large-scale 3D culture to provide a specific biomolecular microenvironment to mimic in vivo mesoderm formation process. After 5 days of spontaneous differentiation period, the resulting 3D tissue constructs are composed of multipotent mesodermal progenitor cells verified by gene and molecular expression profiles. Subsequently the optimal time points to trigger terminal differentiation towards cardiomyogenesis or osteogenesis from the mesodermal tissue constructs is found. A simple and affordable 3D ESC-bioprocess that can reach the scalable production of mesoderm origin tissues with significantly improved correspondent tissue properties is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface modified alginate microcapsules for 3D cell culture

NASA Astrophysics Data System (ADS)

Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

2016-06-01

Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

Development of advanced silicon solar cells for Space Station Freedom

NASA Technical Reports Server (NTRS)

Lillington, David R.

1990-01-01

This report describes the development of large area high efficiency wrapthrough solar cells for Space Station Freedom. The goal of this contract was the development and fabrication of 8 x 8 cm coplanar back contact solar cells with a minimum output of 1.039 watts/cell. The first task in this program was a modeling study to determine the optimum configuration of the cell and to study the effects of surface passivation, substrate resistivity, and back surface field on the BOL and EOL performance. In addition, the optical stack, including the cell cover, AR coatings, and Kapton blanket, was modeled to optimize 'on orbit' operation. The second phase was a manufacturing development phase to develop high volume manufacturing processes for the reliable production of low recombination velocity boron back surface fields, techniques to produce smooth, low leakage wrapthrough holes, passivation, photoresist application methods, and metallization schemes. The final portion of this program was a pilot production phase. Seven hundred solar cells were delivered in this phase. At the end of the program, cells with average efficiencies over 13 percent were being produced with power output in excess of 1.139 watts/cell, thus substantially exceeding the program goal.

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

Bass, H.Z.

As observed from a large panel of mouse T helper clones, there are at least two subsets of CD4{sup +} T cells that both differ in function and demonstrate distinct patterns of cytokine secretion after antigen or mitogen stimulation. Th1 cells synthesize IL-2, INF-{gamma} and lymphotoxin. They produce a DTH reaction in the footpads of naive mice. In addition, Th1 cells are required for the generation of CTL, and they appear to augment IgG2a antibody production. In contrast, by secreting IL-4, IL-5, and IL-6, Th2 cells play an essential role in humoral immunity. TLI consists of high dose, fractionated irradiationmore » delivered selectively to the major lymphoid tissues. Four to six weeks after TLI, the CD4{sup +} cells of the treated mice (counted as a percentage of the total spleen lymphocytes) recover to the similar levels as those in normal BALB/c mice. These CD4{sup +} cells can help normal syngeneic B cells to produce a vigorous antibody response to TNP-KLH in adoptive cell transfer experiments, but the same cells are inactive in the MLR, and they fail to transfer DTH in TNP-KLH primed syngeneic BALB/c mice.« less

Grid-connected polymer solar panels: initial considerations of cost, lifetime, and practicality.

PubMed

Medford, Andrew J; Lilliedal, Mathilde R; Jørgensen, Mikkel; Aarø, Dennis; Pakalski, Heinz; Fyenbo, Jan; Krebs, Frederik C

2010-09-13

Large solar panels were constructed from polymer solar cell modules prepared using full roll-to-roll (R2R) manufacture based on the previously published ProcessOne. The individual flexible polymer solar modules comprising multiple serially connected single cell stripes were joined electrically and laminated between a 4 mm tempered glass window and black Tetlar foil using two sheets of 0.5 mm thick ethylene vinyl acetate (EVA). The panels produced up to 8 W with solar irradiance of ~960 Wm⁻², and had outer dimensions of 1 m x 1.7 m with active areas up to 9180 cm². Panels were mounted on a tracking station and their output was grid connected between testing. Several generations of polymer solar cells and panel constructions were tested in this context to optimize the production of polymer solar panels. Cells lacking a R2R barrier layer were found to degrade due to diffusion of oxygen after less than a month, while R2R encapsulated cells showed around 50% degradation after 6 months but suffered from poor performance due to de-lamination during panel production. A third generation of panels with various barrier layers was produced to optimize the choice of barrier foil and it was found that the inclusion of a thin protective foil between the cell and the barrier foil is critical. The findings provide a preliminary foundation for the production and optimization of large-area polymer solar panels and also enabled a cost analysis of solar panels based on polymer solar cells.

Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors.

PubMed

Haghverdi, Laleh; Lun, Aaron T L; Morgan, Michael D; Marioni, John C

2018-06-01

Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are produced in different laboratories and at different times contain batch effects that may compromise the integration and interpretation of the data. Existing scRNA-seq analysis methods incorrectly assume that the composition of cell populations is either known or identical across batches. We present a strategy for batch correction based on the detection of mutual nearest neighbors (MNNs) in the high-dimensional expression space. Our approach does not rely on predefined or equal population compositions across batches; instead, it requires only that a subset of the population be shared between batches. We demonstrate the superiority of our approach compared with existing methods by using both simulated and real scRNA-seq data sets. Using multiple droplet-based scRNA-seq data sets, we demonstrate that our MNN batch-effect-correction method can be scaled to large numbers of cells.

Cell-based therapeutics from an economic perspective: primed for a commercial success or a research sinkhole?

PubMed

McAllister, Todd N; Dusserre, Nathalie; Maruszewski, Marcin; L'heureux, Nicolas

2008-11-01

Despite widespread hype and significant investment through the late 1980s and 1990s, cell-based therapeutics have largely failed from both a clinical and financial perspective. While the early pioneers were able to create clinically efficacious products, small margins coupled with small initial indications made it impossible to produce a reasonable return on the huge initial investments that had been made to support widespread research activities. Even as US FDA clearance opened up larger markets, investor interest waned, and the crown jewels of cell-based therapeutics went bankrupt or were rescued by corporate bailout. Despite the hard lessons learned from these pioneering companies, many of today's regenerative medicine companies are supporting nearly identical strategies. It remains to be seen whether or not our proposed tenets for investment and commercialization strategy yield an economic success or whether the original model can produce a return on investment sufficient to justify the large up-front investments. Irrespective of which approach yields a success, it is critically important that more of the second-generation products establish profitability if the field is to enjoy continued investment from both public and private sectors.

Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species

PubMed Central

Penketh, Philip G.; Baumann, Raymond P.; Ishiguro, Kimiko; Shyam, Krishnamurthy; Seow, Helen A.; Sartorelli, Alan C.

2010-01-01

Cloretazine [1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]-hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The number of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produce a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line. PMID:18479747

Large-Scale Coherent Vortex Formation in Two-Dimensional Turbulence

NASA Astrophysics Data System (ADS)

Orlov, A. V.; Brazhnikov, M. Yu.; Levchenko, A. A.

2018-04-01

The evolution of a vortex flow excited by an electromagnetic technique in a thin layer of a conducting liquid was studied experimentally. Small-scale vortices, excited at the pumping scale, merge with time due to the nonlinear interaction and produce large-scale structures—the inverse energy cascade is formed. The dependence of the energy spectrum in the developed inverse cascade is well described by the Kraichnan law k -5/3. At large scales, the inverse cascade is limited by cell sizes, and a large-scale coherent vortex flow is formed, which occupies almost the entire area of the experimental cell. The radial profile of the azimuthal velocity of the coherent vortex immediately after the pumping was switched off has been established for the first time. Inside the vortex core, the azimuthal velocity grows linearly along a radius and reaches a constant value outside the core, which agrees well with the theoretical prediction.

In vitro generated Th17 cells support the expansion and phenotypic stability of CD4(+)Foxp3(+) regulatory T cells in vivo.

PubMed

Zhou, Qiong; Hu, Ya; Howard, O M Zack; Oppenheim, Joost J; Chen, Xin

2014-01-01

CD4(+) T cells stimulate immune responses through distinct patterns of cytokine produced by Th1, Th2 or Th17 cells, or inhibit immune responses through Foxp3-expressing regulatory T cells (Tregs). Paradoxically, effector T cells were recently shown to activate Tregs, however, it remains unclear which Th subset is responsible for this effect. In this study, we found that Th17 cells expressed the highest levels of TNF among in vitro generated Th subsets, and most potently promoted expansion and stabilized Foxp3 expression by Tregs when co-transferred into Rag1(-/-) mice. Both TNF and IL-2 produced by Th17 cells contributed to this effect. The stimulatory effect of Th17 cells on Tregs was largely abolished when co-transferred with TNFR2-deficient Tregs. Furthermore, Tregs deficient in TNFR2 also supported a much lower production of IL-17A and TNF expression by co-transferred Th17 cells. Thus, our data indicate that the TNF-TNFR2 pathway plays a crucial role in the reciprocal stimulatory effect of Th17 cells and Tregs. This bidirectional interaction should be taken into account when designing therapy targeting Th17 cells, Tregs, TNF and TNFR2. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

PubMed Central

Jeewandara, Chandima; Adikari, Thiruni N.; Gomes, Laksiri; Fernando, Samitha; Fernando, R. H.; Perera, M. K. T.; Ariyaratne, Dinuka; Kamaladasa, Achala; Salimi, Maryam; Prathapan, Shamini

2015-01-01

Background Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood. Methodology/Principal findings Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus. Conclusions/significance The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials. PMID:25875020

Analysis of Direct Solar Illumination on the Backside of Space Station Solar Cells

NASA Technical Reports Server (NTRS)

Delleur, Ann M.; Kerslake, Thomas W.; Scheiman, David A.

1999-01-01

The International Space Station (ISS) is a complex spacecraft that will take several years to assemble in orbit. During many of the assembly and maintenance procedures, the space station's large solar arrays must he locked, which can significantly reduce power generation. To date, power generation analyses have not included power generation from the backside of the solar cells in a desire to produce a conservative analysis. This paper describes the testing of ISS solar cell backside power generation, analytical modeling and analysis results on an ISS assembly mission.

Nanotopographical control of human osteoprogenitor differentiation.

PubMed

Dalby, Matthew J; Gadegaard, Nikolaj; Curtis, Adam S G; Oreffo, Richard O C

2007-05-01

Current load-bearing orthopaedic implants are produced in 'bio-inert' materials such as titanium alloys. When inserted into the reamed bone during hip or knee replacement surgery the implants interact with mesenchymal populations including the bone marrow. Bio-inert materials are shielded from the body by differentiation of the cells along the fibroblastic lineage producing scar tissue and inferior healing. This is exacerbated by implant micromotion, which can lead to capsule formation. Thus, next-generation implant materials will have to elicit influence over osteoprogenitor differentiation and mesenchymal populations in order to recruit osteoblastic cells and produce direct bone apposition onto the implant. A powerful method of delivering cues to cells is via topography. Micro-scale topography has been shown to affect cell adhesion, migration, cytoskeleton, proliferation and differentiation of a large range of cell types (thus far all cell types tested have been shown to be responsive to topographical cues). More recent research with nanotopography has also shown a broad range of cell response, with fibroblastic cells sensing down to 10 nm in height. Initial studies with human mesenchymal populations and osteoprogenitor populations have again shown strong cell responses to nanofeatures with increased levels of osteocalcin and osteopontin production from the cells on certain topographies. This is indicative of increased osteoblastic activity on the nanotextured materials. Looking at preliminary data, it is tempting to speculate that progenitor cells are, in fact, more responsive to topography than more mature cell types and that they are actively seeking cues from their environment. This review will investigate the range of nanotopographies available to researchers and our present understanding of mechanisms of progenitor cell response. Finally, it will make some speculations of the future of nanomaterials and progenitor cells in tissue engineering.

Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

NASA Technical Reports Server (NTRS)

Crucian, Brian E.; Sams, Clarence F.

2001-01-01

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent distinct monocyte subsets with unique functions. CONCLUSIONS: Whole blood culture eliminates the need to purify cell populations prior to culture and may have Significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. In this study, alterations in cytokine production are demonstrated between whole blood and PBMC activation. It is likely that whole blood activation more accurately represents the in-vivo immune balance than PBMC activation.

Use of the yeast-like cells of Tremella fuciformis as a cell factory to produce a Pleurotus ostreatus hydrophobin.

PubMed

Zhu, Hanyu; Liu, Dongmei; Wang, Yuanyuan; Ren, Danfeng; Zheng, Liesheng; Chen, Liguo; Ma, Aimin

2017-08-01

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens. The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate. Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.

FORSPAN Model Users Guide

USGS Publications Warehouse

Klett, T.R.; Charpentier, Ronald R.

2003-01-01

The USGS FORSPAN model is designed for the assessment of continuous accumulations of crude oil, natural gas, and natural gas liquids (collectively called petroleum). Continuous (also called ?unconventional?) accumulations have large spatial dimensions and lack well defined down-dip petroleum/water contacts. Oil and natural gas therefore are not localized by buoyancy in water in these accumulations. Continuous accumulations include ?tight gas reservoirs,? coalbed gas, oil and gas in shale, oil and gas in chalk, and shallow biogenic gas. The FORSPAN model treats a continuous accumulation as a collection of petroleumcontaining cells for assessment purposes. Each cell is capable of producing oil or gas, but the cells may vary significantly from one another in their production (and thus economic) characteristics. The potential additions to reserves from continuous petroleum resources are calculated by statistically combining probability distributions of the estimated number of untested cells having the potential for additions to reserves with the estimated volume of oil and natural gas that each of the untested cells may potentially produce (total recovery). One such statistical method for combination of number of cells with total recovery, used by the USGS, is called ACCESS.

A Simple and Efficient Method of Slow Freezing for Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

PubMed

Imaizumi, Keitaro; Iha, Momoe; Nishishita, Naoki; Kawamata, Shin; Nishikawa, Shinichi; Akuta, Teruo

2016-01-01

Protocols available for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells are very inefficient and laborious compared to those for the cryopreservation of murine ES/iPS cells or other general cell lines. While the vitrification method may be adequate when working with small numbers of human ES/iPS cells, it requires special skills and is unsuitable when working with large cell numbers. Here, we describe a simple and efficient method for the cryopreservation of hES/hiPS cells that is based on a conventional slow freezing method that uses a combination of Pronase/EDTA for Stem™ and CP-5E™ [final concentrations: 6 % hydroxyethyl starch, 5 % DMSO, and 5 % ethylene glycol in saline]. CP-5E™ is highly effective for the cryopreservation of small cell clumps produced by hES/hiPS colony detachment in the presence of Pronase and EDTA (Pronase/EDTA for Stem™, a formulation containing multiple digestive enzymes from Streptomyces griseus). This novel method would be quite useful for large-scale hES/iPS cell banking for use in clinical applications.

Feasibility studies of oncornavirus production in microcarrier cultures.

PubMed

Manousos, M; Ahmed, M; Torchio, C; Wolff, J; Shibley, G; Stephens, R; Mayyasi, S

1980-06-01

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

PDT-treated apoptotic cells induce macrophage synthesis NO

NASA Astrophysics Data System (ADS)

Song, S.; Xing, D.; Zhou, F. F.; Chen, W. R.

2009-11-01

Nitric oxide (NO) is a biologically active molecule which has multi-functional in different species. As a second messenger and neurotransmitter, NO is not only an important regulatory factor between cells' information transmission, but also an important messenger in cell-mediated immunity and cytotoxicity. On the other side, NO is involving in some diseases' pathological process. In pathological conditions, the macrophages are activated to produce a large quantity of nitric oxide synthase (iNOS), which can use L-arginine to produce an excessive amount of NO, thereby killing bacteria, viruses, parasites, fungi, tumor cells, as well as in other series of the immune process. In this paper, photofrin-based photodynamic therapy (PDT) was used to treat EMT6 mammary tumors in vitro to induce apoptotic cells, and then co-incubation both apoptotic cells and macrophages, which could activate macrophage to induce a series of cytotoxic factors, especially NO. This, in turn, utilizes macrophages to activate a cytotoxic response towards neighboring tumor cells. These results provided a new idea for us to further study the immunological mechanism involved in damaging effects of PDT, also revealed the important function of the immune effect of apoptotic cells in PDT.

Preliminary Evaluation of Glyceric Acid-producing Ability of Acidomonas methanolica NBRC104435 from Glycerol Containing Methanol.

PubMed

Sato, Shun; Kitamoto, Dai; Habe, Hiroshi

2017-06-01

Some acetic acid bacteria produce large amounts of glyceric acid (GA) from glycerol in culture broth. However, methanol, which is a major contaminant of raw glycerol derived from the biodiesel fuel industry, sharply decreases cell growth and GA production [AMB Express, 3, 20, 2013]. Thus, we evaluated the methylotrophic acetic acid bacterium Acidomonas methanolica NBRC104435 for its ability to produce GA from glycerol containing methanol. This strain accumulated GA in its culture broth when 1-3 wt% glycerol was available as a carbon source. We observed improved cell growth and GA accumulation when 1 vol% methanol was added to the 3-5 wt% glycerol medium. The maximum concentration of GA was 12.8 g/L in medium containing 3 wt% glycerol plus 1 vol% methanol. In addition, the enantiomeric excess (ee) of the GA produced was revealed to be 44%, indicating that this strain converted glycerol to d-GA with a lower enantioselectivity than other acetic acid bacteria, which had 70-99% ee.

Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945.

PubMed

Mitsunaga, Hitoshi; Meissner, Lena; Palmen, Thomas; Bamba, Takeshi; Büchs, Jochen; Fukusaki, Eiichiro

2016-04-01

Poly(γ-glutamic acid) (PGA) is a polymer composed of L- and/or D-glutamic acids that is produced by Bacillus sp. Because the polymer has various features as water soluble, edible, non-toxic and so on, it has attracted attention as a candidate for many applications such as foods, cosmetics and so on. However, although it is well known that the intracellular metabolism of Bacillus sp. is mainly regulated by catabolite control, the effect of the catabolite control on the PGA producing Bacillus sp. is largely unknown. This study is the first report of metabolome analysis on the PGA producing Bacillus sp. that reveals the effect of carbon catabolite control on the metabolism of PGA producing Bacillus licheniformis ATCC 9945. Results showed that the cells cultivated in glycerol-containing medium showed higher PGA production than the cells in glucose-containing medium. Furthermore, metabolome analysis revealed that the activators of CcpA and CodY, global regulatory proteins of the intracellular metabolism, accumulated in the cells cultivated in glycerol-containing and glucose-containing medium, respectively, with CodY apparently inhibiting PGA production. Moreover, the cells seemed to produce glutamate from citrate and ammonium using glutamine synthetase/glutamate synthase. Pulsed addition of di-ammonium hydrogen citrate, as suggested by the metabolome result, was able to achieve the highest value so far for PGA production in B. licheniformis. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes.

PubMed

Xu, Yong-Nan; Uhm, Sang-Jun; Koo, Bon-Chul; Kwon, Mo-Sun; Roh, Ji-Yeol; Yang, Jung-Seok; Choi, Hyun-Yong; Heo, Young-Tae; Cui, Xiang-Shun; Yoon, Joon-Ho; Ko, Dae-Hwan; Kim, Teoan; Kim, Nam-Hyung

2013-01-20

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein (EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5%±2.2% v.s. 22.9%±2.9%). Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer (NT). Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells. Copyright © 2013. Published by Elsevier Ltd.

Environmental enrichment alters dentate granule cell morphology in oldest-old rat.

PubMed

Darmopil, Sanja; Petanjek, Zdravko; Mohammed, Abdul H; Bogdanović, Nenad

2009-08-01

The hippocampus of aged rats shows marked age-related morphological changes that could cause memory deficits. Experimental evidence has established that environmental enrichment attenuates memory deficits in aged rats. We therefore studied whether environmental enrichment produces morphological changes on the dentate granule cells of aged rats. Fifteen male Sprague-Dawley rats, 24 months of age, were randomly distributed in two groups that were housed under standard (n = 7) or enriched (n = 8) environmental conditions for 26 days. Quantitative data of dendritic morphology from dentate gyrus granule cells were obtained on Golgi-Cox stained sections. Environmental enrichment significantly increased the complexity and size of dendritic tree (total number of segments increased by 61% and length by 116%), and spine density (88% increase). There were large interindividual differences within the enriched group, indicating differential individual responses to environmental stimulation. Previous studies in young animals have shown changes produced by environmental enrichment in the morphology of dentate gyrus granule cells. The results of the present study show that environmental enrichment can also produce changes in dentate granule cell morphology in the senescent brain. In conclusion, the hippocampus retains its neuroplastic capacity during aging, and enriched environmental housing conditions can attenuate age-related dendritic regression and synaptic loss, thus preserving memory functions.

Allelopathic effects of volatile monoterpenoids produced by Salvia leucophylla: Inhibition of cell proliferation and DNA synthesis in the root apical meristem of Brassica campestris seedlings.

PubMed

Nishida, Nami; Tamotsu, Satoshi; Nagata, Noriko; Saito, Chieko; Sakai, Atsushi

2005-05-01

Salvia leucophylla, a shrub observed in coastal south California, produces several volatile monoterpenoids (camphor, 1,8-cineole, beta-pinene, alpha-pinene, and camphene) that potentially act as allelochemicals. The effects of these were examined using Brassica campestris as the test plant. Camphor, 1,8-cineole, and beta-pinene inhibited germination of B. campestris seeds at high concentrations, whereas alpha-pinene and camphene did not. Root growth was inhibited by all five monoterpenoids in a dose-dependent manner, but hypocotyl growth was largely unaffected. The monoterpenoids did not alter the sizes of matured cells in either hypocotyls or roots, indicating that cell expansion is relatively insensitive to these compounds. They did not decrease the mitotic index in the shoot apical region, but specifically lowered mitotic index in the root apical meristem. Moreover, morphological and biochemical analyses on the incorporation of 5-bromo-2'-deoxyuridine into DNA demonstrated that the monoterpenoids inhibit both cell-nuclear and organelle DNA synthesis in the root apical meristem. These results suggest that the monoterpenoids produced by S. leucophylla could interfere with the growth of other plants in its vicinity through inhibition of cell proliferation in the root apical meristem.

Artificial cloning of domestic animals

PubMed Central

Keefer, Carol L.

2015-01-01

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770

Artificial cloning of domestic animals.

PubMed

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

Single Cell Oil Production from Hydrolysates of Inulin by a Newly Isolated Yeast Papiliotrema laurentii AM113 for Biodiesel Making.

PubMed

Wang, Guangyuan; Liu, Lin; Liang, Wenxing

2018-01-01

Microbial oils are among the most attractive alternative feedstocks for biodiesel production. In this study, a newly isolated yeast strain, AM113 of Papiliotrema laurentii, was identified as a potential lipid producer, which could accumulate a large amount of intracellular lipids from hydrolysates of inulin. P. laurentii AM113 was able to produce 54.6% (w/w) of intracellular oil in its cells and 18.2 g/l of dry cell mass in a fed-batch fermentation. The yields of lipid and biomass were 0.14 and 0.25 g per gram of consumed sugar, respectively. The lipid productivity was 0.092 g of oil per hour. Compositions of the fatty acids produced were C 14:0 (0.9%), C 16:0 (10.8%), C 16:1 (9.7%), C 18:0 (6.5%), C 18:1 (60.3%), and C 18:2 (11.8%). Biodiesel obtained from the extracted lipids could be burnt well. This study not only provides a promising candidate for single cell oil production, but will also probably facilitate more efficient biodiesel production.

Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

PubMed Central

Purtha, Whitney E.; Tedder, Thomas F.; Johnson, Syd

2011-01-01

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) persist after clearance of infection, yet the specific and nonredundant role MBCs play in subsequent protection is unclear. After resolution of West Nile virus infection in mice, we demonstrate that LLPCs were specific for a single dominant neutralizing epitope, such that immune serum poorly inhibited a variant virus that encoded a mutation at this critical epitope. In contrast, a large fraction of MBC produced antibody that recognized both wild-type (WT) and mutant viral epitopes. Accordingly, antibody produced by the polyclonal pool of MBC neutralized WT and variant viruses equivalently. Remarkably, we also identified MBC clones that recognized the mutant epitope better than the WT protein, despite never having been exposed to the variant virus. The ability of MBCs to respond to variant viruses in vivo was confirmed by experiments in which MBCs were adoptively transferred or depleted before secondary challenge. Our data demonstrate that class-switched MBC can respond to variants of the original pathogen that escape neutralization of antibody produced by LLPC without a requirement for accumulating additional somatic mutations. PMID:22162833

Using hollow carbon nanospheres as a light-induced free radical generator to overcome chemotherapy resistance.

PubMed

Wang, Liming; Sun, Qiang; Wang, Xin; Wen, Tao; Yin, Jun-Jie; Wang, Pengyang; Bai, Ru; Zhang, Xiang-Qian; Zhang, Lu-Hua; Lu, An-Hui; Chen, Chunying

2015-02-11

Under evolutionary pressure from chemotherapy, cancer cells develop resistance characteristics such as a low redox state, which eventually leads to treatment failures. An attractive option for combatting resistance is producing a high concentration of produced free radicals in situ. Here, we report the production and use of dispersible hollow carbon nanospheres (HCSs) as a novel platform for delivering the drug doxorubicine (DOX) and generating additional cellular reactive oxygen species using near-infrared laser irradiation. These irradiated HCSs catalyzed sufficiently persistent free radicals to produce a large number of heat shock factor-1 protein homotrimers, thereby suppressing the activation and function of resistance-related genes. Laser irradiation also promoted the release of DOX from lysosomal DOX@HCSs into the cytoplasm so that it could enter cell nuclei. As a result, DOX@HCSs reduced the resistance of human breast cancer cells (MCF-7/ADR) to DOX through the synergy among photothermal effects, increased generation of free radicals, and chemotherapy with the aid of laser irradiation. HCSs can provide a unique and versatile platform for combatting chemotherapy-resistant cancer cells. These findings provide new clinical strategies and insights for the treatment of resistant cancers.

Properties of solar generators with reflectors and radiators

NASA Astrophysics Data System (ADS)

Ebeling, W. D.; Rex, D.; Bierfischer, U.

1980-06-01

Radiation cooled concentrator systems using silicon and GaAs cells were studied. The principle of radiation cooling by the reflector surfaces is discussed for cylindrical parabolic reflectors (SARA), truncated hexagonal pyramids, and a small trough configuration. Beam paths, collection properties for imperfect orientation, and thermal optimization parameters were analyzed. The three concentrating systems with radiation cooling offer advantages over the plane panel and over the large trough. With silicon solar cells they exhibit considerably lower solar cell consumption per Kw and also lower mass per kW. With GaAs cells the SARA system reduces the number of solar cells needed per kW to less than 10%. Also in all other cases SARA offers the best values for alpha and F sub sol, as long as narrow angular tolerances of the panel orientation can be met. Analysis of the energy collecting properties for imperfect orientation shows the superiority of the hexagonal concentrator. This device can produce power for even large angles between the sun and the panel normal.

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

PubMed

Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

2018-01-01

Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.

Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

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

Kanakura, Y.; Kuriu, A.; Waki, N.

Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared,more » suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast.« less

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

Production and Assessment of Decellularized Pig and Human Lung Scaffolds

PubMed Central

Niles, Jean; Riddle, Michael; Vargas, Gracie; Schilagard, Tuya; Ma, Liang; Edward, Kert; La Francesca, Saverio; Sakamoto, Jason; Vega, Stephanie; Ogadegbe, Marie; Mlcak, Ronald; Deyo, Donald; Woodson, Lee; McQuitty, Christopher; Lick, Scott; Beckles, Daniel; Melo, Esther; Cortiella, Joaquin

2013-01-01

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production. PMID:23638920

Production and assessment of decellularized pig and human lung scaffolds.

PubMed

Nichols, Joan E; Niles, Jean; Riddle, Michael; Vargas, Gracie; Schilagard, Tuya; Ma, Liang; Edward, Kert; La Francesca, Saverio; Sakamoto, Jason; Vega, Stephanie; Ogadegbe, Marie; Mlcak, Ronald; Deyo, Donald; Woodson, Lee; McQuitty, Christopher; Lick, Scott; Beckles, Daniel; Melo, Esther; Cortiella, Joaquin

2013-09-01

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.

Effects of Chitosan on Candida albicans: Conditions for Its Antifungal Activity

PubMed Central

Peña, Antonio; Sánchez, Norma Silvia; Calahorra, Martha

2013-01-01

The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 μg·mL−1 produced (a) an efflux of K+ and stimulation of extracellular acidification, (b) an inhibition of Rb+ uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca2+. It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K+, it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca2+, (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL−1 are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells. PMID:23844364

Organization of testicular interstitial tissue of an Australian rodent, the spinifex hopping mouse, Notomys alexis.

PubMed

Peirce, E J; Breed, W G

1990-05-01

The organization of testicular interstitial tissue of the spinifex hopping mouse, Notomys alexis differs from that of other rodents. It comprises between 10.3% and 17.3% (average 15.0%) of the total testicular volume, and is variable in its organization both at different locations within the testis of the one animal and among different individuals. Abundant, closely packed Leydig cells are usually present; however, in some regions large, thick-walled blood vessels and extensive peritubular lymphatic spaces, often lacking an endothelium adjacent to the Leydig cells, are also prominent. The Leydig cells in contact with the large blood vessels and lymphatics, unlike those in regions where lymph is sparse, are not densely packed and sometimes contain numerous lipid droplets. Ultrastructure of Leydig cells is typical of steroid-producing cells; however, mitochondria are often extremely large, unusual in shape or bizarrely arranged in relation to one another. Also electron-dense bodies displaying a paracrystalline-like internal structure of parallel, electron-dense filaments arranged in a lattice pattern occur in the cytoplasm of many cells. The significance of these unusual ultrastructural features and the organization of the interstitial tissue remain to be determined conclusively, but may relate to steroid synthesis, secretion and uptake.

An FGF-driven feed-forward circuit patterns the cardiopharyngeal mesoderm in space and time

PubMed Central

Razy-Krajka, Florian; Gravez, Basile; Kaplan, Nicole; Racioppi, Claudia; Wang, Wei

2018-01-01

In embryos, multipotent progenitors divide to produce distinct progeny and express their full potential. In vertebrates, multipotent cardiopharyngeal progenitors produce second-heart-field-derived cardiomyocytes, and branchiomeric skeletal head muscles. However, the mechanisms underlying these early fate choices remain largely elusive. The tunicate Ciona emerged as an attractive model to study early cardiopharyngeal development at high resolution: through two asymmetric and oriented divisions, defined cardiopharyngeal progenitors produce distinct first and second heart precursors, and pharyngeal muscle (aka atrial siphon muscle, ASM) precursors. Here, we demonstrate that differential FGF-MAPK signaling distinguishes between heart and ASM precursors. We characterize a feed-forward circuit that promotes the successive activations of essential ASM determinants, Hand-related, Tbx1/10 and Ebf. Finally, we show that coupling FGF-MAPK restriction and cardiopharyngeal network deployment with cell divisions defines the timing of gene expression and permits the emergence of diverse cell types from multipotent progenitors. PMID:29431097

Production and characterization of vaccines based on flaviviruses defective in replication

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

Mason, Peter W.; Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019; Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019

2006-08-01

To develop new vaccine candidates for flavivirus infections, we have engineered two flaviviruses, yellow fever virus (YFV) and West Nile virus (WNV), that are deficient in replication. These defective pseudoinfectious viruses (PIVs) lack a functional copy of the capsid (C) gene in their genomes and are incapable of causing spreading infection upon infection of cells both in vivo and in vitro. However, they produce extracellular E protein in form of secreted subviral particles (SVPs) that are known to be an effective immunogen. PIVs can be efficiently propagated in trans-complementing cell lines making high levels of C or all three viralmore » structural proteins. PIVs derived from YFV and WNV, demonstrated very high safety and immunization produced high levels of neutralizing antibodies and protective immune response. Such defective flaviviruses can be produced in large scale under low biocontainment conditions and should be useful for diagnostic or vaccine applications.« less

Dynamics of storage reserve deposition during Brassica rapa L. pollen and seed development in microgravity

NASA Technical Reports Server (NTRS)

Kuang, A.; Popova, A.; McClure, G.; Musgrave, M. E.

2005-01-01

Pollen and seeds share a developmental sequence characterized by intense metabolic activity during reserve deposition before drying to a cryptobiotic form. Neither pollen nor seed development has been well studied in the absence of gravity, despite the importance of these structures in supporting future long-duration manned habitation away from Earth. Using immature seeds (3-15 d postpollination) of Brassica rapa L. cv. Astroplants produced on the STS-87 flight of the space shuttle Columbia, we compared the progress of storage reserve deposition in cotyledon cells during early stages of seed development. Brassica pollen development was studied in flowers produced on plants grown entirely in microgravity on the Mir space station and fixed while on orbit. Cytochemical localization of storage reserves showed differences in starch accumulation between spaceflight and ground control plants in interior layers of the developing seed coat as early as 9 d after pollination. At this age, the embryo is in the cotyledon elongation stage, and there are numerous starch grains in the cotyledon cells in both flight and ground control seeds. In the spaceflight seeds, starch was retained after this stage, while starch grains decreased in size in the ground control seeds. Large and well-developed protein bodies were observed in cotyledon cells of ground control seeds at 15 d postpollination, but their development was delayed in the seeds produced during spaceflight. Like the developing cotyledonary tissues, cells of the anther wall and filaments from the spaceflight plants contained numerous large starch grains, while these were rarely seen in the ground controls. The tapetum remained swollen and persisted to a later developmental stage in the spaceflight plants than in the ground controls, even though most pollen grains appeared normal. These developmental markers indicate that Brassica seeds and pollen produced in microgravity were physiologically younger than those produced in 1 g. We hypothesize that microgravity limits mixing of the gaseous microenvironments inside the closed tissues and that the resulting gas composition surrounding the seeds and pollen retards their development.

Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

PubMed

Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

2008-10-14

High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

PubMed Central

Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

2008-01-01

High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a α-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins. PMID:18840687

IL-27 triggers IL-10 production in Th17 cells via a c-Maf/RORγt/Blimp-1 signal to promote the progression of endometriosis

PubMed Central

Chang, Kai-Kai; Liu, Li-Bing; Jin, Li-Ping; Zhang, Bing; Mei, Jie; Li, Hui; Wei, Chun-Yan; Zhou, Wen-Jie; Zhu, Xiao-Yong; Shao, Jun; Li, Da-Jin; Li, Ming-Qing

2017-01-01

Endometriosis is an estrogen-dependent inflammatory disease. The anti-inflammatory cytokine IL-10 is also increased in endometriosis. IL-10 production by Th17 cells is critical for limiting autoimmunity and inflammatory responses. However, the mechanism of inducing IL-10-producing Th17 cells is still largely unknown. The present study investigated the differentiation mechanism and role of IL-10-producing Th17 cells in endometriosis. Here, we report that IL-10+Th17 cells are significantly increased in the peritoneal fluid of women with endometriosis, along with an elevation of IL-27, IL-6 and TGF-β. Compared with peripheral CD4+ T cells, endometrial CD4+ T cells highly expressed IL-27 receptors, especially the ectopic endometrium. Under external (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and local (estrogen, IL-6 and TGF-β) environmental regulation, IL-27 from macrophages and endometrial stromal cells (ESCs) induces IL-10 production in Th17 cells in vitro and in vivo. This process may be mediated through the interaction between c-musculoaponeurotic fibrosarconna (c-Maf) and retinoic acid-related orphan receptor gamma t (RORγt), and associated with the upregulation of downstream B lymphocyte-induced maturation protein-1 (Blimp-1). IL-10+Th17 cells, in turn, stimulate the proliferation and implantation of ectopic lesions and accelerate the progression of endometriosis. These results suggest that IL-27 is a pivotal regulator in endometriotic immune tolerance by triggering Th17 cells to produce IL-10 and promoting the rapid growth and implantation of ectopic lesions. This finding provides a scientific basis for potential therapeutic strategies aimed at preventing the development of endometriosis, especially for patients with high levels of IL-10+Th17 cells. PMID:28300844

Glucose-dependent insulinotropic polypeptide-producing K cells in dexamethasone-treated rats.

PubMed

Koko, V; Glisic, R; Todorovic, V; Drndarevic, N; Mitrovic, O

2008-12-01

Some studies indicate that diabetes mellitus exerts an influence on the gastrointestinal tract and its diffuse neuroendocrine system (DNES) in regard to cellular density and neuroendocrine content. Since there is no data about relationship between experimentally induced non-insulin-dependent (type 2) diabetes mellitus (NIDDM) on the gut K cells, the aim of our study was to investigate immunohistochemical, stereological and ultrastructural changes of rat K cells after 12 days of dexamethasone treatment. Twenty male Wistar rats aged 30 days were given daily intraperitoneally 2 mg kg(-1) dexamethasone (group DEX, 10 rats) or saline (group C, 10 rats) for 12 days. Tissue specimens were obtained from each antrum with corpus and different parts of the small (SI) and large intestine (LI) of all animals. Immunohistochemistry was carried out using antisera against the GIP and insulin. Transmission electron microscopy was also used. Although, according to the literature data, rat K cells are present in the duodenum and jejunum and, to a lesser extent, in the ileum, in the present study we observed that those cells were abundant also in all parts of the LI. We observed generally that GIP-producing K cells were augmented in all parts of SI and decreased in the LI of DEX rats. Insulin immunoreactivity (ir) coexpressed with GIP-ir in K cells and was stronger in the SI of DEX rats as compared with C rats. We also found by electron microscopy that small intestinal K cells have features not only of GIP-secreted but also of insulin-secreted cells. We concluded that dexamethasone treatment caused proliferation of K cells in the rat SI, and simultaneously transformation of GIP-producing K cells to insulin-synthesizing cells.

Visualizing the molecular sociology at the HeLa cell nuclear periphery.

PubMed

Mahamid, Julia; Pfeffer, Stefan; Schaffer, Miroslava; Villa, Elizabeth; Danev, Radostin; Cuellar, Luis Kuhn; Förster, Friedrich; Hyman, Anthony A; Plitzko, Jürgen M; Baumeister, Wolfgang

2016-02-26

The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed the native structure and organization of the cytoplasmic translation machinery. Analysis of a large dynamic structure-the nuclear pore complex-revealed variations detectable at the level of individual complexes. Cryo-ET was used to visualize previously elusive structures, such as nucleosome chains and the filaments of the nuclear lamina, in situ. Elucidation of the lamina structure provides insight into its contribution to metazoan nuclear stiffness. Copyright © 2016, American Association for the Advancement of Science.

Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes.

PubMed

Lü, Shuanghong; Liu, Sheng; He, Wenjun; Duan, Cuimi; Li, Yanmin; Liu, Zhiqiang; Zhang, Ye; Hao, Tong; Wang, Yanmeng; Li, Dexue; Wang, Changyong; Gao, Shaorong

2008-09-01

Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.

Extracting Silicon From Sodium-Process Products

NASA Technical Reports Server (NTRS)

Kapur, V.; Sanjurjo, A.; Sancier, K. M.; Nanis, L.

1982-01-01

New acid leaching process purifies silicon produced in reaction between silicon fluoride and sodium. Concentration of sodium fluoride and other impurities and byproducts remaining in silicon are within acceptable ranges for semi-conductor devices. Leaching process makes sodium reduction process more attractive for making large quantities of silicon for solar cells.

Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

USDA-ARS?s Scientific Manuscript database

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

ASSESSMENT OF PC12 CELL DIFFERENTIATION AND NEURITE GROWTH: A COMPARISON OF MORPHOLOGICAL AND NEUROCHEMICAL MEASURES.

EPA Science Inventory

In order to screen large numbers of chemicals for their potential to produce developmental neurotoxicity new, in vitro methods are needed. One approach is to develop methods based on the biologic processes which underlie brain development including the growth and maturation of ce...

Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds.

PubMed

Mohanty, Soumyaranjan; Larsen, Layla Bashir; Trifol, Jon; Szabo, Peter; Burri, Harsha Vardhan Reddy; Canali, Chiara; Dufva, Marin; Emnéus, Jenny; Wolff, Anders

2015-10-01

One of the major challenges in producing large scale engineered tissue is the lack of ability to create large highly perfused scaffolds in which cells can grow at a high cell density and viability. Here, we explore 3D printed polyvinyl alcohol (PVA) as a sacrificial mould in a polymer casting process. The PVA mould network defines the channels and is dissolved after curing the polymer casted around it. The printing parameters determined the PVA filament density in the sacrificial structure and this density resulted in different stiffness of the corresponding elastomer replica. It was possible to achieve 80% porosity corresponding to about 150 cm(2)/cm(3) surface to volume ratio. The process is easily scalable as demonstrated by fabricating a 75 cm(3) scaffold with about 16,000 interconnected channels (about 1m(2) surface area) and with a channel to channel distance of only 78 μm. To our knowledge this is the largest scaffold ever to be produced with such small feature sizes and with so many structured channels. The fabricated scaffolds were applied for in-vitro culturing of hepatocytes over a 12-day culture period. Smaller scaffolds (6×4 mm) were tested for cell culturing and could support homogeneous cell growth throughout the scaffold. Presumably, the diffusion of oxygen and nutrient throughout the channel network is rapid enough to support cell growth. In conclusion, the described process is scalable, compatible with cell culture, rapid, and inexpensive. Copyright © 2015. Published by Elsevier B.V.

Anonymous sources: where do adult β cells come from?

PubMed Central

German, Michael S.

2013-01-01

Evidence that the pool of insulin-producing β cells in the pancreas is reduced in both major forms of diabetes mellitus has led to efforts to understand β cell turnover in the adult pancreas. Unfortunately, previous studies have reached opposing conclusions regarding the source of new β cells during regeneration in the adult pancreas. In this issue of the JCI, Xiao et al. use a novel mouse model for detecting new β cells derived from non–β cells to demonstrate the absence of β cell neogenesis from non–β cells during normal postnatal growth and in models of β cell regeneration. This work adds to mounting evidence that in most physiological and pathological conditions, β cell neogenesis may not make large contributions to the postnatal β cell pool — at least not in rodents. PMID:23619356

Production and characterization of alginate microcapsules produced by a vibrational encapsulation device.

PubMed

Mazzitelli, S; Tosi, A; Balestra, C; Nastruzzi, C; Luca, G; Mancuso, F; Calafiore, R; Calvitti, M

2008-09-01

The optimization, through a Design of Experiments (DoE) approach, of a microencapsulation procedure for isolated neonatal porcine islets (NPI) is described. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An alginate/polyornithine encapsulation procedure, developed and validated in our laboratory for almost a decade, was used to embody pancreatic islets. We analyzed different experimental parameters including frequency of vibration, amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. We produced calcium-alginate gel microbeads with excellent morphological characteristics as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology, viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

JPRS Report, Science & Technology, Japan, Government and Private Sector Joint R&D Projects

DTIC Science & Technology

1988-08-16

is that if this system uses exogenous genes represented by the DNA that codes for hepatitis B surface antigen, polyvaccines can be produced easily...that can express in large quantities molecular clones of genetic products is awaited. Vectors using cells of higher mammals including man have been...functions of IVM and MMD using cultured spinal nerve cells with a great advantage that the same sample can be used for both electrophysiological and

Umbelliprenin is cytotoxic against QU-DB large cell lung cancer cell line but anti-proliferative against A549 adenocarcinoma cells

PubMed Central

2012-01-01

Background Umbelliprenin is a natural compound, belonging to the class of sesquiterpene coumarins. Recently, umbelliprenin has attracted the researchers' attention for its antitumor activities against skin tumors. Its effect on lung cancer is largely unknown. The aim of our study was to investigate the effects of this natural compound, which is expected to have low adverse effects, on lung cancer. Methods The QU-DB large cell and A549 adenocarcinoma lung cancer cell lines were treated with umbelliprenin. IC50 values were estimated using methyl thiazolely diphenyl-tetrazolium bromide (MTT) assay, in which a decrease in MTT reduction can occur as a result of cell death or cell proliferation inhibition. To quantify the rate of cell death at IC50 values, flow cytometry using Annexin V-FITC (for apoptotic cells), and propidium iodide (for necrotic cells) dyes were employed. Results Data from three independent MTT experiments in triplicate revealed that IC50 values for QU-DB and A549 were 47 ± 5.3 μM and 52 ± 1.97 μM, respectively. Annexin V/PI staining demonstrated that umbelliprenin treatment at IC50 induced 50% cell death in QU-DB cells, but produced no significant death in A549 cells until increasing the umbelliprenin concentration to IC80. The pattern of cell death was predominantly apoptosis in both cell lines. When peripheral blood mononuclear cells were treated with 50 μM and less concentrations of umbelliprenin, no suppressive effect was observed. Conclusions We found cytotoxic/anti-proliferative effects of umbelliprenin against two different types of lung cancer cell lines. PMID:23351548

Induction of polyclonal antibody synthesis by human allogeneic and autologous helper factors

PubMed Central

1979-01-01

Human helper factors were obtained from supernates of 48 h unidirectional allogeneic and autologous mixed lymphocyte reactions. These supernates were shown to induce the production of large amounts of immunoglobulin by tonsillar and peripheral blood mononuclear cells. Abundant polyclonal activation to antibody production occurred in these cultures in the absence of antigenic challenge which was similar in degree to that produced by pokeweed mitogen. This was documented by quantitating plasma cells, specific plaque-forming cells, and secreted immunoglobulin. In addition, the supplementation of companion cultures with sheep erythrocytes resulted in a significant enhancement of the specific plaque-forming cell response without an appreciable change in plasma cell number of secreted Ig. PMID:156242

Genomic localization of the Z/EG transgene in the mouse genome.

PubMed

Colombo, Sophie; Kumasaka, Mayuko; Lobe, Corrinne; Larue, Lionel

2010-02-01

The Z/EG transgenic mouse line, produced by Novak et al., displays tissue-specific EGFP expression after Cre-mediated recombination. The autofluorescence of EGFP allows the visualization of cells of interest displaying Cre recombination. The initial construct was designed such that cells without Cre recombination express the beta-galactosidase marker, facilitating counterselection. We used inverse PCR to identify the site of integration of the Z/EG transgene, to improve the efficiency of homozygous Z/EG mouse production. Recombined cells produced large amounts of EGFP protein, resulting in higher levels of fluorescence and therefore greater contrast with nonrecombined cells. We mapped the transgene to the G1 region of chromosome 5. This random insertion was found to have occurred 230-bp upstream from the start codon of the Rasa4 gene. The insertion of the Z/EG transgene in the C57BL/6 genetic background had no effect on Rasa4 expression. Homozygous Z/EG mice therefore had no obvious phenotype. (c) 2009 Wiley-Liss, Inc.

Process for producing large grain cadmium telluride

DOEpatents

Hasoon, Falah S.; Nelson, Art J.

1996-01-01

A process for producing a cadmium telluride polycrystalline film having grain sizes greater than about 20 .mu.m. The process comprises providing a substrate upon which cadmium telluride can be deposited and placing that substrate within a vacuum chamber containing a cadmium telluride effusion cell. A polycrystalline film is then deposited on the substrate through the steps of evacuating the vacuum chamber to a pressure of at least 10.sup.-6 torr.; heating the effusion cell to a temperature whereat the cell releases stoichiometric amounts of cadmium telluride usable as a molecular beam source for growth of grains on the substrate; heating the substrate to a temperature whereat a stoichiometric film of cadmium telluride can be deposited; and releasing cadmium telluride from the effusion cell for deposition as a film on the substrate. The substrate then is placed in a furnace having an inert gas atmosphere and heated for a sufficient period of time at an annealing temperature whereat cadmium telluride grains on the substrate grow to sizes greater than about 20 .mu.m.

Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.

PubMed

Jo, Junghyun; Xiao, Yixin; Sun, Alfred Xuyang; Cukuroglu, Engin; Tran, Hoang-Dai; Göke, Jonathan; Tan, Zi Ying; Saw, Tzuen Yih; Tan, Cheng-Peow; Lokman, Hidayat; Lee, Younghwan; Kim, Donghoon; Ko, Han Seok; Kim, Seong-Oh; Park, Jae Hyeon; Cho, Nam-Joon; Hyde, Thomas M; Kleinman, Joel E; Shin, Joo Heon; Weinberger, Daniel R; Tan, Eng King; Je, Hyunsoo Shawn; Ng, Huck-Hui

2016-08-04

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells

PubMed Central

Winter, Mark R.; Liu, Mo; Monteleone, David; Melunis, Justin; Hershberg, Uri; Goderie, Susan K.; Temple, Sally; Cohen, Andrew R.

2015-01-01

Summary Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex. PMID:26344906

On-the-fly selection of cell-specific enhancers, genes, miRNAs and proteins across the human body using SlideBase

PubMed Central

Ienasescu, Hans; Li, Kang; Andersson, Robin; Vitezic, Morana; Rennie, Sarah; Chen, Yun; Vitting-Seerup, Kristoffer; Lagoni, Emil; Boyd, Mette; Bornholdt, Jette; de Hoon, Michiel J. L.; Kawaji, Hideya; Lassmann, Timo; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Carninci, Piero; Sandelin, Albin

2016-01-01

Genomics consortia have produced large datasets profiling the expression of genes, micro-RNAs, enhancers and more across human tissues or cells. There is a need for intuitive tools to select subsets of such data that is the most relevant for specific studies. To this end, we present SlideBase, a web tool which offers a new way of selecting genes, promoters, enhancers and microRNAs that are preferentially expressed/used in a specified set of cells/tissues, based on the use of interactive sliders. With the help of sliders, SlideBase enables users to define custom expression thresholds for individual cell types/tissues, producing sets of genes, enhancers etc. which satisfy these constraints. Changes in slider settings result in simultaneous changes in the selected sets, updated in real time. SlideBase is linked to major databases from genomics consortia, including FANTOM, GTEx, The Human Protein Atlas and BioGPS. Database URL: http://slidebase.binf.ku.dk PMID:28025337

Solar cell anomaly detection method and apparatus

NASA Technical Reports Server (NTRS)

Miller, Emmett L. (Inventor); Shumka, Alex (Inventor); Gauthier, Michael K. (Inventor)

1981-01-01

A method is provided for detecting cracks and other imperfections in a solar cell, which includes scanning a narrow light beam back and forth across the cell in a raster pattern, while monitoring the electrical output of the cell to find locations where the electrical output varies significantly. The electrical output can be monitored on a television type screen containing a raster pattern with each point on the screen corresponding to a point on the solar cell surface, and with the brightness of each point on the screen corresponding to the electrical output from the cell which was produced when the light beam was at the corresponding point on the cell. The technique can be utilized to scan a large array of interconnected solar cells, to determine which ones are defective.

Innate-like behavior of human invariant natural killer T cells during herpes simplex virus infection.

PubMed

Novakova, Lucie; Nevoralova, Zuzana; Novak, Jan

2012-01-01

Invariant natural killer T (iNKT) cells, CD1d restricted T cells, are involved in the immune responses against various infection agents. Here we describe their behavior during reactivation of human herpes simplex virus (HSV). iNKT cells exhibit only discrete changes, which however, reached statistically significant level due to the relatively large patient group. Higher percentage of iNKT cells express NKG2D. iNKT cells down-regulate NKG2A in a subset of patients. Finally, iNKT cells enhance their capacity to produce TNF-α. Our data suggests that iNKT cells are involved in the immune response against HSV and contribute mainly to its early, innate phase. Copyright © 2012 Elsevier Inc. All rights reserved.

Sexy males and sexless females: the origin of triploid apomicts.

PubMed

Muralidhar, P; Haig, D

2017-05-01

Apomixis and polyploidy are closely associated in angiosperms, but the evolutionary reason for this association is unknown. Taraxacum officinale, the common dandelion, exists both as diploid sexuals and triploid apomicts. Here, in the context of T. officinale, we provide a model of the evolution of triploid apomicts from diploid sexuals. We posit an apomictic allele that arrests female meiosis in diploids, so that the plant produces diploid egg cells that can develop without fertilization, but haploid pollen. We propose occasional fertilization of diploid egg cells by haploid pollen, resulting in triploid apomicts that produce triploid egg cells but largely nonfunctional pollen. The irreversibility of this process renders diploid partial apomicts evolutionarily short-lived, and results in fixation of triploid apomicts except when they suffer extreme selective disadvantages. Our model can account for the high genetic diversity found in T. officinale triploid populations, because recombinant haploid pollen produced by diploids allows the apomictic allele to spread onto many genetic backgrounds. This leads to multiple clonal lineages in the newly apomictic population, and thereby alleviates some of the usual pitfalls of asexual reproduction.

Development of a high efficiency thin silicon solar cell

NASA Technical Reports Server (NTRS)

Storti, G.; Culik, J.; Wrigley, C.

1980-01-01

Significant improvements in open-circuit voltage and conversion efficiency, even on relatively high bulk resistivity silicon, were achieved by using a screen-printed aluminum paste back surface field. A 4 sq cm 50 micron m thick cell was fabricated from textured 10 omega-cm silicon which had an open-circuit voltage of 595 mV and AMO conversion efficiency at 25 C of 14.3%. The best 4 sq cm 50 micron thick cell (2 omega-cm silicon) produced had an open-circuit voltage of 607 mV and an AMO conversion efficiency of 15%. Processing modifications are described which resulted in better front contact integrity and reduced breakage. These modifications were utilized in the thin cell pilot line to fabricate 4 sq cm cells with an average AMO conversion efficiency at 25 C of better than 12.5% and with lot yields as great as 51% of starts; a production rate of 10,000 cells per month was demonstrated. A pilot line was operated which produced large area (25 cm) ultra-thin cells with an average AMO conversion efficiency at 25 deg of better than 11.5% and a lot yield as high as 17%.

Advances in Large Grain Resonators for the European XFEL

NASA Astrophysics Data System (ADS)

Singer, W.; Aderhold, S.; Iversen, J.; Kreps, G.; Matheisen, A.; Singer, X.; Twarowski, K.; Weise, H.; Pekeler, M.; Scholz, F.; Spaniol, B.; Stiedl, E.

2011-03-01

An overview of the activities within the DESY test program of 1.3 GHz TESLA shape 9-cell Large Grain (LG) resonators for the European XFEL, which have taken place in last 4 years, is presented. Attention is devoted to development of LG disc production and cavity fabrication from this material, focusing in particular on aspects of production at reasonable accuracy and costs. More than 200 LG discs were manufactured, eleven 9-cell resonators produced, partially treated at the company Research Instruments (RI) (former ACCEL) and finally treated and RF tested at DESY. Two of the LG cavities are currently used in the FLASH accelerator operation, which is the best demonstration of the feasibility of the LG application. The program compares large grain material with standard sheet niobium. Some data and perspectives of the LG application are discussed.

Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

PubMed Central

Blackstone, Britani N.; Palmer, Andre F.; Rilo, Horacio R.

2014-01-01

Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development. PMID:24410263

Synthesis and cytotoxic evaluation of some new 4(3H)-quinazolinones on HeLa cell line

PubMed Central

Khodarahmi, G.A.; Shamshiri, M.; Hassanzadeh, F.

2012-01-01

Quinazolinone backbone is present in a large number of bioactive substances. Since remarkable cytotoxic activity is associated with some 4(3H)-quinazolinones, in this study some 4(3H)-quinazolinone were synthesized and screened against HeLa cells. The synthesis was performed via reaction of anthranilic acid with dicarboxylic anhydrides to produce carboxylic acids derivatives. The products were heated in acetic anhydride to produce benzoxazinones. Finally, 4(3H)-quinazolinones were synthesized by reaction between benzoxazinones and primary amines. The assessment of the structure of the synthesized compounds was based on spectral data (FT-IR, Mass and 1HNMR). Subsequently, cytotoxic activity of compounds 3, 6, 9 and 13 (individually and in combination with doxorubicin) was evaluated on HeLa cell line using MTT assay. The results indicated that the tested compounds did not show significant cytotoxicity alone and in combination with doxorubicin (1 and 20 μM). PMID:23181089

Eradication of Large Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a Single Cancer-Specific Point Mutation.

PubMed

Leisegang, Matthias; Engels, Boris; Schreiber, Karin; Yew, Poh Yin; Kiyotani, Kazuma; Idel, Christian; Arina, Ainhoa; Duraiswamy, Jaikumar; Weichselbaum, Ralph R; Uckert, Wolfgang; Nakamura, Yusuke; Schreiber, Hans

2016-06-01

Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS. By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734-43. ©2015 AACRSee related commentary by Liu, p. 2602. ©2015 American Association for Cancer Research.

Large-scale generation of cell-derived nanovesicles

NASA Astrophysics Data System (ADS)

Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim, S. C.; Gho, Y. S.; Park, J.

2014-09-01

Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

Advances in Large Grain/Single Crystal SC Resonators at DESY

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

W. Singer; A. Brinkmann; A. Ermakov

The main aim of the DESY large grain R&D program is to check whether this option is reasonable to apply for fabrication of ca. 1'000 XFEL cavities. Two aspects are being pursued. On one hand the basic material investigation, on the other hand the material availability, fabrication and preparation procedure. Several single cell large grain cavities of TESLA shape have been fabricated and tested. The best accelerating gradients of 41 MV/m was measured on electropolished cavity. First large grain nine-cell cavities worldwide have been produced under contract of DESY with ACCEL Instruments Co. All three cavities fulfil the XFEL specificationmore » already in first RF test after only BCP (Buffered Chemical Polishing) treatment and 800 degrees C annealing. Accelerating gradient of 27 - 29 MV/m was reached. A fabrication method of single crystal cavity of ILC like shape was proposed. A single cell single crystal cavity was build at the company ACCEL. Accelerating gradient of 37.5 MV/m reached after only 112 microns BCP and in situ baking 120 degrees C for 6 hrs with the quality factor higher as 2x1010. The developed method can be extended on fabrication of multi cell single crystal cavities.« less

Cytokine-induced killer cells co-cultured with dendritic cells loaded with the protein lysate produced by radiofrequency ablation induce a specific antitumor response

PubMed Central

SHAN, CHAN-CHAN; SHI, LIANG-RONG; DING, MEI-QIAN; ZHU, YI-BEI; LI, XIAO-DONG; XU, BIN; JIANG, JING-TING; WU, CHANG-PING

2015-01-01

Radiofrequency ablation (RFA) causes coagulative necrosis of tumor tissue and the production of local tumor protein debris. These fragments of tumor protein debris contain a large number of various antigens, which can stimulate a specific cellular immune response. In the present study, dendritic cells (DCs) were loaded with tumor protein lysate antigens that were produced in situ by RFA, and were used to treat murine colon carcinoma in combination with cytokine-induced killer (CIK) cells. Subsequent to the treatment of murine colon carcinoma by RFA, the in situ supernatant of tumor lysis was collected and the DCs were loaded with the lysate antigen to generate Ag-DCs. CIK cells induced from the spleen cells of mice were co-cultured with Ag-DCs to generate Ag-DC-CIK cells. The results revealed that the Ag-DC-CIK cells exhibited strong antitumor activity in vitro and in vivo. The morphology and immunophenotypes of these cells were determined using microscopy and flow cytometry, respectively. The cytotoxic activity of Ag-DC-CIK cells was determined using a CCK-8 assay. To establish a mouse model, mice were randomized into Ag-DC-CIK, DC-CIK, CIK and PBS control groups and monitored for tumor growth and survival time. ANOVA was used to compare the trends in the three groups for implanted tumor volumes. The log-rank test was used to compare the survival time. The present findings indicated that DCs loaded with the protein lysate antigens of tumors, produced in situ by RFA, combined with CIK cells may be a novel strategy for cancer treatment. PMID:25788999

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos.

PubMed

Murakami, M; Ferguson, C E; Perez, O; Boediono, A; Paccamonti, D; Bondioli, K R; Godke, R A

2006-01-01

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

PubMed Central

McDermott, Martina; Eustace, Alex J.; Busschots, Steven; Breen, Laura; Crown, John; Clynes, Martin; O’Donovan, Norma; Stordal, Britta

2014-01-01

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance. PMID:24639951

The in vitro release of cytokines and growth factors from fibrin membranes produced through horizontal centrifugation.

PubMed

Lourenço, Emanuelle Stellet; Mourão, Carlos Fernando de Almeida Barros; Leite, Paulo Emílio Corrêa; Granjeiro, José Mauro; Calasans-Maia, Mônica Diuana; Alves, Gutemberg Gomes

2018-05-01

Platelet-rich fibrin membranes are biomaterials widely used for therapeutic purposes, and canonically produced through the processing of peripheral blood with fixed-angle rotor centrifuges. In this work, we evaluate the in vitro stability and release of cytokines and growth factors when these biomaterials are produced with a horizontal swing-out clinical centrifuge. Membranes produced from the blood of 14 donors were morphologically evaluated by scanning electron microscopy and fluorescence microscopy, and their stability was assessed by photographic recording after incubation in culture medium for up to 28 days. The release of 27 cytokines and growth factors was monitored for three weeks through a multiparametric immunoassay. The fibrin membranes presented complex three-dimensional structure with a high density of nucleated cells. A large release of growth factors [platelet derived growth factor, fibroblastic growth factor (bFGF), and vascular endothelial growth factor] was detected in the first 24 h, followed by time-dependent decay, maintaining significant concentrations after three weeks. Both anti-inflammatory and pro-inflammatory cytokines presented different release peaks, maintaining high rates of elution for up to 21 days. Chemokines of relevance in tissue repair [RANTES, granulocyte colony-stimulating factor (G-CSF)] were also produced in large quantities throughout the experimental period. The present results demonstrate that blood-derived fibrin membranes with high structural stability and cell content can be generated by horizontal centrifugation, being able of a prolonged production/release of growth factors and pro- and anti-inflammatory cytokines. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1373-1380, 2018. © 2018 Wiley Periodicals, Inc.

Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells.

PubMed

Petit, Laetitia; Gibert, Maryse; Gourch, Abdelkader; Bens, Marcelle; Vandewalle, Alain; Popoff, Michel R

2003-03-01

Epsilon toxin is produced by Clostridium perfringens types B and D which are responsible for fatal intestinal diseases in animals. The main biological activity of epsilon toxin is the production of oedema in various organs. We have previously found that epsilon toxin forms a large membrane complex in MDCK cells which is not internalized into cell, and induces cell volume enlargement and loss of cell viability (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., Popoff, M. R. (1997) J Bacteriol 179, 6480-6487). Here, we show that epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low-molecular-weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide and finally to the loss of cell viability.

Spermatogonial stem cells from domestic animals: progress and prospects.

PubMed

Zheng, Yi; Zhang, Yaqing; Qu, Rongfeng; He, Ying; Tian, Xiue; Zeng, Wenxian

2014-03-01

Spermatogenesis, an elaborate and male-specific process in adult testes by which a number of spermatozoa are produced constantly for male fertility, relies on spermatogonial stem cells (SSCs). As a sub-population of undifferentiated spermatogonia, SSCs are capable of both self-renewal (to maintain sufficient quantities) and differentiation into mature spermatozoa. SSCs are able to convert to pluripotent stem cells during in vitro culture, thus they could function as substitutes for human embryonic stem cells without ethical issues. In addition, this process does not require exogenous transcription factors necessary to produce induced-pluripotent stem cells from somatic cells. Moreover, combining genetic engineering with germ cell transplantation would greatly facilitate the generation of transgenic animals. Since germ cell transplantation into infertile recipient testes was first established in 1994, in vivo and in vitro study and manipulation of SSCs in rodent testes have been progressing at a staggering rate. By contrast, their counterparts in domestic animals, despite the failure to reach a comparable level, still burgeoned and showed striking advances. This review outlines the recent progressions of characterization, isolation, in vitro propagation, and transplantation of spermatogonia/SSCs from domestic animals, thereby shedding light on future exploration of these cells with high value, as well as contributing to the development of reproductive technology for large animals.

Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation.

PubMed Central

Peña, A; Ramírez, J; Rosas, G; Calahorra, M

1995-01-01

The internal pH of yeast cells was determined by measuring the fluorescence changes of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), which was introduced into the cells by electroporation. This may be a suitable procedure for the following reasons. (i) Only minor changes in the physiological status of the cells seemed to be produced. (ii) The dye did not seem to leak at a significant rate from the cells. (iii) Different incubation conditions produced large fluorescence changes in the dye, which in general agree with present knowledge of the proton movements of the yeast cell under different conditions. (iv) Pyranine introduced by electroporation seemed to be located in the cytoplasm and to avoid the vacuole, and therefore it probably measured actual cytoplasmic pH. (v) Correction factors to obtain a more precise estimation of the internal pH are not difficult to apply, and the procedure may be useful for other yeasts and microorganisms, as well as for the introduction of other substances into cells. Values for the cytoplasmic pHs of yeast cells that were higher than those reported previously were obtained, probably because this fluorescent indicator did not seem to penetrate into the cell vacuole. PMID:7860582

Techno-Economic Analysis of Scalable Coal-Based Fuel Cells

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

Chuang, Steven S. C.

Researchers at The University of Akron (UA) have demonstrated the technical feasibility of a laboratory coal fuel cell that can economically convert high sulfur coal into electricity with near zero negative environmental impact. Scaling up this coal fuel cell technology to the megawatt scale for the nation’s electric power supply requires two key elements: (i) developing the manufacturing technology for the components of the coal-based fuel cell, and (ii) long term testing of a kW scale fuel cell pilot plant. This project was expected to develop a scalable coal fuel cell manufacturing process through testing, demonstrating the feasibility of buildingmore » a large-scale coal fuel cell power plant. We have developed a reproducible tape casting technique for the mass production of the planner fuel cells. Low cost interconnect and cathode current collector material was identified and current collection was improved. In addition, this study has demonstrated that electrochemical oxidation of carbon can take place on the Ni anode surface and the CO and CO 2 product produced can further react with carbon to initiate the secondary reactions. One important secondary reaction is the reaction of carbon with CO 2 to produce CO. We found CO and carbon can be electrochemically oxidized simultaneously inside of the anode porous structure and on the surface of anode for producing electricity. Since CH 4 produced from coal during high temperature injection of coal into the anode chamber can cause severe deactivation of Ni-anode, we have studied how CH 4 can interact with CO 2 to produce in the anode chamber. CO produced was found able to inhibit coking and allow the rate of anode deactivation to be decreased. An injection system was developed to inject the solid carbon and coal fuels without bringing air into the anode chamber. Five planner fuel cells connected in a series configuration and tested. Extensive studies on the planner fuels and stack revealed that the planner fuel cell stack is not suitable for operation with carbon and coal fuels due to lack of mechanical strength and difficulty in sealing. We have developed scalable processes for manufacturing of process for planner and tubular cells. Our studies suggested that tubular cell stack could be the only option for scaling up the coal-based fuel cell. Although the direct feeding of coal into fuel cell can significantly simplify the fuel cell system, the durability of the fuel cell needs to be further improved before scaling up. We are developing a tubular fuel cell stack with a coal injection and a CO 2 recycling unit.« less

Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells.

PubMed

Shin, Dong-Jun; Park, Ji-Yun; Jang, Youn-Young; Lee, Je-Jung; Lee, Youn-Kyung; Shin, Myung-Geun; Jung, Ji-Youn; Carson, William E; Cho, Duck; Kim, Sang-Ki

2013-06-15

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3(+)CD8(+) TCRαβ(-)TCRγδ(-)CD4(-)CD21(-)CD11c(+/-)CD11d(+/-)CD44(+). The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Influence of variable milk quality premiums on observed milk quality.

PubMed

Nightingale, C; Dhuyvetter, K; Mitchell, R; Schukken, Y

2008-03-01

The objective of this study was to evaluate a premium program for very high quality milk in a US cooperative. Data were available on a monthly basis from a large US milk cooperative from April 1998 through December 2005. The data set consisted of 36,930 observations representing producer-months. The actual amount of the low bulk tank somatic cell count (BTSCC) premium varied from $0.15 per hundred pounds (cwt.) of milk to $1.00/cwt. with steps in between of $0.50 and $0.60 per cwt. of milk during the data collection period. Data analysis was done to evaluate the impact of the premium program on average BTSCC and on the probability of a producer to ship milk with <100,000 cells/mL in a given month. The results showed a strong effect of the premium program on both the average BTSCC and the probability of producing milk with very low BTSCC. On average, the BTSCC of all the milk in the cooperative was reduced by 22,000 cells during the high premium period. The probability of producing milk with BTSCC <100,000 doubled during some months of the high premium period from 4 to 8%, and an associated 10% increase in probability to produce milk below 200,000 cells/mL was observed. The data clearly indicate that premium offerings for very high quality milk affect the overall milk quality in the population affected by the premium. Producers responded to the high premiums and the overall impact on milk quality was substantial. We argue that the combination of a penalty program for high BTSCC milk with a premium program for very high quality milk (low BTSCC) provides a strong incentive for improvement of milk quality.

Concomitant differentiation of a population of mouse embryonic stem cells into neuron-like cells and Schwann cell-like cells in a slow-flow microfluidic device

PubMed Central

Ramamurthy, Poornapriya; White, Joshua B.; Park, Joong Yull; Hume, Richard I.; Ebisu, Fumi; Mendez, Flor; Takayama, Shuichi; Barald, Kate F

2016-01-01

Background To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining Spiral Ganglion Neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived “neurons” could potentially substitute for lost or damaged SGN. mESC-derived “Schwann cells” produce MIF as do all Schwann cells (Huang et al., 2002; Roth et al., 2007, 2008) and could attract SGN to “ cell coated” implant. Results Neuron- and Schwann cell-like cells were produced from a common population of mESC in an ultra-slow flow microfluidic device. As the populations interacted; “neurons” grew over the “Schwann cell” lawn and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing “Schwann cells” were used to “coat” a CI: mouse SGN and MIF-induced “neurons” grew directionally to the CI and to a wild type but not MIF-knock out Organ of Corti explant. Conclusions Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. PMID:27761977

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

NASA Image and Video Library

1992-09-20

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

TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network

PubMed Central

Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L.; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2017-01-01

Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae. In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. PMID:28325812

TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network.

PubMed

Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2017-05-05

Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. Copyright © 2017 Usaj et al.

The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals.

PubMed

Decotto, Eva; Spradling, Allan C

2005-10-01

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains "cyst progenitor" stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. We now show, by lineage marking, that the ovarian niche also contains a second type of stem cell. These "escort stem cells" morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, we show that JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms.

High rate lithium/thionyl chloride bipolar battery development

NASA Technical Reports Server (NTRS)

Russell, Philip G.; Goebel, F.

1994-01-01

Presented in viewgraph format are results and accomplishments on the development of lithium/thionyl chloride bipolar batteries. Results include the development of manufacturing capability for producing large quantities of uniform cathodes and bipolar plates; the development of assembly, sealing, and activation procedures for fabrication of battery modules containing up to 150 cells in bipolar configuration; and the successful demonstration of a 10.7 kW 150-cell module with constant power pulse discharge, 20 second pulse, and 10 percent duty cycle.

Superantigens Modulate Bacterial Density during Staphylococcus aureus Nasal Colonization

PubMed Central

Xu, Stacey X.; Kasper, Katherine J.; Zeppa, Joseph J.; McCormick, John K.

2015-01-01

Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization. PMID:26008236

Polyunsaturated Aldehydes from Large Phytoplankton of the Atlantic Ocean Surface (42°N to 33°S)

PubMed Central

Bartual, Ana; Arandia-Gorostidi, Néstor; Cózar, Andrés; Morillo-García, Soledad; Ortega, María Jesús; Vidal, Montserrat; Cabello, Ana María; González-Gordillo, Juan Ignacio; Echevarría, Fidel

2014-01-01

Polyunsaturated aldehydes (PUAs) are organic compounds mainly produced by diatoms, after cell wounding. These compounds are increasingly reported as teratogenic for species of grazers and deleterious for phytoplanktonic species, but there is still scarce information regarding concentration ranges and the composition of PUAs in the open ocean. In this study, we analyzed the spatial distribution and the type of aldehydes produced by the large-sized (>10 μm) phytoplankton in the Atlantic Ocean surface. Analyses were conducted on PUAs released after mechanical disruption of the phytoplankton cells, referred to here as potential PUAs (pPUAs). Results show the ubiquitous presence of pPUA in the open ocean, including upwelling areas, as well as oligotrophic gyres. Total pPUA concentrations ranged from zero to 4.18 pmol from cells in 1 L. Identified PUAs were heptadienal, octadienal and decadienal, with heptadienal being the most common (79% of total stations). PUA amount and composition across the Atlantic Ocean was mainly related to the nitrogen:phosphorus ratio, suggesting nutrient-driven mechanisms of PUA production. Extending the range of trophic conditions considered by adding data reported for productive coastal waters, we found a pattern of PUA variation in relation to trophic status. PMID:24473169

Spatial and temporal dynamics of cellulose degradation and biofilm formation by Caldicellulosiruptor obsidiansis and Clostridium thermocellum Caldicellulosiruptor obsidiansis

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

Wang, Zhiwu; Lee, Sueng-Hwan; Elkins, James G

2011-01-01

Cellulose degradation is one of the major bottlenecks of a consolidated bioprocess that employs cellulolytic bacterial cells as catalysts to produce biofuels from cellulosic biomass. In this study, we investigated the spatial and temporal dynamics of cellulose degradation by Caldicellulosiruptor obsidiansis, which does not produce cellulosomes, and Clostridium thermocellum, which does produce cellulosomes. Results showed that the degradation of either regenerated or natural cellulose was synchronized with biofilm formation, a process characterized by the formation and fusion of numerous crater-like depressions on the cellulose surface. In addition, the dynamics of biofilm formation were similar in both bacteria, regardless of cellulosomemore » production. Only the areas of cellulose surface colonized by microbes were significantly degraded, highlighting the essential role of the cellulolytic biofilm in cellulose utilization. After initial attachment, the microbial biofilm structure remained thin, uniform and dense throughout the experiment. A cellular automaton model, constructed under the assumption that the attached cells divide and produce daughter cells that contribute to the hydrolysis of the adjacent cellulose, can largely simulate the observed process of biofilm formation and cellulose degradation. This study presents a model, based on direct observation, correlating cellulolytic biofilm formation with cellulose degradation.« less

Disorganized Steroidogenesis in Adrenocortical Carcinoma, a Case Study.

PubMed

Uchida, Toyoyoshi; Nishimoto, Koshiro; Fukumura, Yuki; Asahina, Miki; Goto, Hiromasa; Kawano, Yui; Shimizu, Fumitaka; Tsujimura, Akira; Seki, Tsugio; Mukai, Kuniaki; Kabe, Yasuaki; Suematsu, Makoto; Gomez-Sanchez, Celso E; Yao, Takashi; Horie, Shigeo; Watada, Hirotaka

2017-03-01

Most adrenocortical carcinomas (ACCs) produce excessive amounts of steroid hormones including aldosterone, cortisol, and steroid precursors. However, aldosterone- and cortisol-producing cells in ACCs have not yet been immunohistochemically described. We present a case of ACC causing mild primary aldosteronism and subclinical Cushing's syndrome. Removal of the tumor cured both conditions. In order to examine the expression patterns of the steroidogenic enzymes responsible for adrenocortical hormone production, 10 tumor portions were immunohistochemically analyzed for aldosterone synthase (CYP11B2), 11β-hydroxylase (CYP11B1, cortisol-synthesizing enzyme), 3β-hydroxysteroid dehydrogenase (3βHSD, upstream enzyme for both CYP11B2 and CYP11B1), and 17α-hydroxylase/C17-20 lyase (CYP17, upstream enzyme for CYP11B1, but not for CYP11B1). CYP11B2, CYP11B1, and 3βHSD were expressed sporadically, and their expression patterns varied significantly among the different tumor portions examined. The expression of these enzymes was random and not associated with each other. CYP17 was expressed throughout the tumor, even in CYP11B2-positive cells. Small tumor cell populations were aldosterone- or cortisol-producing cells, as judged by 3βHSD coinciding with either CYP11B2 or CYP11B1, respectively. These results suggest that the tumor produced limited amounts of aldosterone and cortisol due to the lack of the coordinated expression of steroidogenic enzymes, which led to mild clinical expression in this case. We delineated the expression patterns of steroidogenic enzymes in ACC. The coordinated expression of steroidogenic enzymes in normal and adenoma cells was disturbed in ACC cells, resulting in the inefficient production of steroid hormones in relation to the large tumor volume.

Combination of automated high throughput platforms, flow cytometry, and hierarchical clustering to detect cell state.

PubMed

Kitsos, Christine M; Bhamidipati, Phani; Melnikova, Irena; Cash, Ethan P; McNulty, Chris; Furman, Julia; Cima, Michael J; Levinson, Douglas

2007-01-01

This study examined whether hierarchical clustering could be used to detect cell states induced by treatment combinations that were generated through automation and high-throughput (HT) technology. Data-mining techniques were used to analyze the large experimental data sets to determine whether nonlinear, non-obvious responses could be extracted from the data. Unary, binary, and ternary combinations of pharmacological factors (examples of stimuli) were used to induce differentiation of HL-60 cells using a HT automated approach. Cell profiles were analyzed by incorporating hierarchical clustering methods on data collected by flow cytometry. Data-mining techniques were used to explore the combinatorial space for nonlinear, unexpected events. Additional small-scale, follow-up experiments were performed on cellular profiles of interest. Multiple, distinct cellular profiles were detected using hierarchical clustering of expressed cell-surface antigens. Data-mining of this large, complex data set retrieved cases of both factor dominance and cooperativity, as well as atypical cellular profiles. Follow-up experiments found that treatment combinations producing "atypical cell types" made those cells more susceptible to apoptosis. CONCLUSIONS Hierarchical clustering and other data-mining techniques were applied to analyze large data sets from HT flow cytometry. From each sample, the data set was filtered and used to define discrete, usable states that were then related back to their original formulations. Analysis of resultant cell populations induced by a multitude of treatments identified unexpected phenotypes and nonlinear response profiles.

A generic, cost-effective, and scalable cell lineage analysis platform

PubMed Central

Biezuner, Tamir; Spiro, Adam; Raz, Ofir; Amir, Shiran; Milo, Lilach; Adar, Rivka; Chapal-Ilani, Noa; Berman, Veronika; Fried, Yael; Ainbinder, Elena; Cohen, Galit; Barr, Haim M.; Halaban, Ruth; Shapiro, Ehud

2016-01-01

Advances in single-cell genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells. Current sequencing-based methods for cell lineage analysis depend on low-resolution bulk analysis or rely on extensive single-cell sequencing, which is not scalable and could be biased by functional dependencies. Here we show an integrated biochemical-computational platform for generic single-cell lineage analysis that is retrospective, cost-effective, and scalable. It consists of a biochemical-computational pipeline that inputs individual cells, produces targeted single-cell sequencing data, and uses it to generate a lineage tree of the input cells. We validated the platform by applying it to cells sampled from an ex vivo grown tree and analyzed its feasibility landscape by computer simulations. We conclude that the platform may serve as a generic tool for lineage analysis and thus pave the way toward large-scale human cell lineage discovery. PMID:27558250

Genetic modification of cells for transplantation.

PubMed

Lai, Yi; Drobinskaya, Irina; Kolossov, Eugen; Chen, Chunguang; Linn, Thomas

2008-01-14

Progress in gene therapy has produced promising results that translate experimental research into clinical treatment. Gene modification has been extensively employed in cell transplantation. The main barrier is an effective gene delivery system. Several viral vectors were utilized in end-stage differentiated cells. Recently, successful applications were described with adenovirus-associated vectors. As an alternative, embryonic stem cell- and stem cell-like systems were established for generation of tissue-specified gene-modified cells. Owing to the feasibility for genetic manipulations and the self-renewing potency of these cells they can be used in a way enabling large-scale in vitro production. This approach offers the establishment of in vitro cell culture systems that will deliver sufficient amounts of highly purified, immunoautologous cells suitable for application in regenerative medicine. In this review, the current technology of gene delivery systems to cells is recapitulated and the latest developments for cell transplantation are discussed.

Extraterrestrial processing and manufacturing of large space systems. Volume 3: Executive summary

NASA Technical Reports Server (NTRS)

Miller, R. H.; Smith, D. B. S.

1979-01-01

Facilities and equipment are defined for refining processes to commercial grade of lunar material that is delivered to a 'space manufacturing facility' in beneficiated, primary processed quality. The manufacturing facilities and the equipment for producing elements of large space systems from these materials and providing programmatic assessments of the concepts are also defined. In-space production processes of solar cells (by vapor deposition) and arrays, structures and joints, conduits, waveguides, RF equipment radiators, wire cables, converters, and others are described.

Construction of a Food Grade Recombinant Bacillus subtilis Based on Replicative Plasmids with an Auxotrophic Marker for Biotransformation of d-Fructose to d-Allulose.

PubMed

He, Weiwei; Mu, Wanmeng; Jiang, Bo; Yan, Xin; Zhang, Tao

2016-04-27

A food grade recombinant Bacillus subtilis that produces d-psicose 3-epimerase (DPEase; EC 5.1.3.30) was constructed by transforming a replicative multicopy plasmid with a d-alanine racemase gene marker into B. subtilis 1A751 with the d-alanine racemase gene knocked out. The DPEase was expressed in B. subtilis without antibiotic resistance genes and without adding antibiotics during fermentation. Whole cells of the food grade recombinant B. subtilis were used to biotransform d-fructose to d-allulose. The two tandem promoters, including the HpaII and P43 promoters, increased expression levels compared to the use of one promoter, HpaII. For large-scale d-allulose production, the optimal enzyme dose was 40 enzyme activity units of dry cells per gram of d-fructose, which produced a 28.5% turnover yield in 60 min. The recombinant plasmid exhibited stability over 100 generations. This food grade recombinant B. subtilis may be used for large-scale d-allulose production in the food industry.

Directed evolution of enzymes using microfluidic chips

NASA Astrophysics Data System (ADS)

Pilát, Zdeněk.; Ježek, Jan; Šmatlo, Filip; Kaůka, Jan; Zemánek, Pavel

2016-12-01

Enzymes are highly versatile and ubiquitous biological catalysts. They can greatly accelerate large variety of reactions, while ensuring appropriate catalytic activity and high selectivity. These properties make enzymes attractive biocatalysts for a wide range of industrial and biomedical applications. Over the last two decades, directed evolution of enzymes has transformed the field of protein engineering. We have devised microfluidic systems for directed evolution of haloalkane dehalogenases in emulsion droplets. In such a device, individual bacterial cells producing mutated variants of the same enzyme are encapsulated in microdroplets and supplied with a substrate. The conversion of a substrate by the enzyme produced by a single bacterium changes the pH in the droplet which is signalized by pH dependent fluorescence probe. The droplets with the highest enzymatic activity can be separated directly on the chip by dielectrophoresis and the resultant cell lineage can be used for enzyme production or for further rounds of directed evolution. This platform is applicable for fast screening of large libraries in directed evolution experiments requiring mutagenesis at multiple sites of a protein structure.

Replication of the Apparent Excess Heat Effect in a Light Water-Potassium Carbonate-Nickel Electrolytic Cell

NASA Technical Reports Server (NTRS)

Niedra, Janis M.; Myers, Ira T.; Fralick, Gustave C.; Baldwin, Richard S.

1996-01-01

Replication of experiments claiming to demonstrate excess heat production in light water-Ni-K2CO3 electrolytic cells was found to produce an apparent excess heat of 11 W maximum, for 60 W electrical power into the cell. Power gains range from 1.06 to 1.68. The cell was operated at four different dc current levels plus one pulsed current run at 1 Hz, 10% duty cycle. The 28 liter cell used in these verification tests was on loan from a private corporation whose own tests with similar cells are documented to produce 50 W steady excess heat for a continuous period exceeding hundreds of days. The apparent excess heat can not be readily explained either in terms of nonlinearity of the cell's thermal conductance at a low temperature differential or by thermoelectric heat pumping. However, the present data do admit efficient recombination of dissolved hydrogen-oxygen as an ordinary explanation. Calorimetry methods and heat balance calculations for the verification tests are described. Considering the large magnitude of benefit if this effect is found to be a genuine new energy source, a more thorough investigation of evolved heat in the nickel-hydrogen system in both electrolytic and gaseous loading cells remains warranted.

Examination of the physical properties of Microcystis aeruginosa flocs produced on coagulation with metal salts.

PubMed

Gonzalez-Torres, A; Putnam, J; Jefferson, B; Stuetz, R M; Henderson, R K

2014-09-01

Coagulation-flocculation (C-F) is a key barrier to cyanobacterial and algal cell infiltration in water treatment plants during seasonal blooms. However, the resultant cell floc properties, in terms of size, strength and density, which dominate under different coagulation conditions and govern cell removal, are not well understood. This paper investigated the floc properties produced during C-F of the cyanobacterium, Microcystis aeruginosa, under low and high doses of aluminium sulphate and ferric chloride coagulants and at different pH values, so as to promote charge neutralisation (CN) and sweep flocculation (SF) dominant conditions (or a combination of these). It was demonstrated that application of ferric chloride produced larger flocs that resulted in higher cell removal during jar testing. These flocs were also larger than those observed for natural organic matter (NOM) and kaolin, suggesting a role of algogenic organic matter (AOM) as an inherent bioflocculant. Under SF conditions, stronger flocs were produced; however, these had lower capacity for size recovery after exposure to high shear. Analysis of particle size distribution demonstrated that large scale fragmentation followed by erosion dominated for CN while erosion dominated under SF conditions. Overall, marked differences were observed dependent on the coagulation regime imposed that have implications for improving robustness of cell removal by downstream separation processes. While the cyanobacterium, M. aeruginosa, appeared to share general floc characteristics commonly observed for NOM and kaolin flocs, there were distinct differences in terms of size and strength, which may be attributed to AOM. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hypoxia promotes production of neural crest cells in the embryonic head.

PubMed

Scully, Deirdre; Keane, Eleanor; Batt, Emily; Karunakaran, Priyadarssini; Higgins, Debra F; Itasaki, Nobue

2016-05-15

Hypoxia is encountered in either pathological or physiological conditions, the latter of which is seen in amniote embryos prior to the commencement of a functional blood circulation. During the hypoxic stage, a large number of neural crest cells arise from the head neural tube by epithelial-to-mesenchymal transition (EMT). As EMT-like cancer dissemination can be promoted by hypoxia, we investigated whether hypoxia contributes to embryonic EMT. Using chick embryos, we show that the hypoxic cellular response, mediated by hypoxia-inducible factor (HIF)-1α, is required to produce a sufficient number of neural crest cells. Among the genes that are involved in neural crest cell development, some genes are more sensitive to hypoxia than others, demonstrating that the effect of hypoxia is gene specific. Once blood circulation becomes fully functional, the embryonic head no longer produces neural crest cells in vivo, despite the capability to do so in a hypoxia-mimicking condition in vitro, suggesting that the oxygen supply helps to stop emigration of neural crest cells in the head. These results highlight the importance of hypoxia in normal embryonic development. © 2016. Published by The Company of Biologists Ltd.

Large numbers of interleukins-22- and -17A-producing T helper cells in cholangiocarcinoma related to liver fluke infection.

PubMed

Su, Si-Biao; Zhang, Jian-Feng; Huang, Fei-Fei; Cen, Yu; Jiang, Hai-Xing

2017-08-01

Cholangiocarcinoma (CCA) associated with liver fluke infection involves inflammatory and immune processes; however, whether these involve the proinflammatory cytokine IL-17A and proliferative cytokine IL-22 remains unclear. Here, numbers of IL-22- and IL-17A-producing Th cells and cytokine concentrations in 30 patients with CCA and long-term liver fluke infection, 40 patients with liver-fluke infection but not CCA, and 16 healthy controls were compared. Analyses were performed using immunohistochemistry, flow cytometry, ELISA and RT-PCR. Immunohistochemical staining showed weaker expression of IL-22 and IL-17A in patients with CCA with than in those without liver fluke infection (P < 0.01). Flow cytometry revealed significantly greater median proportions of IL-22-producing T helper cells in patients with CCA (2.2%) than in those without it (0.69%) or controls (0.4%, P < 0.001). Similar results were obtained for IL-17A-producing T helper cells. ELISA revealed plasma concentrations of IL-22 were 1.3-fold higher in patients with CCA than in those without it and 4.6-fold higher than in controls (P < 0.001). Plasma concentrations of IL-17A were 2.5-fold higher in patients with CCA than in those without it, and 21-fold higher than in controls (P < 0.001). Amounts of IL-22 and IL-17A mRNAs in blood were significantly higher in patients with CCA than in the other two groups. Proportions of CD4 + CD45RO + T cells producing IL-22 correlated with proportions producing IL-17A (r = 0.759; P < 0.001), and plasma concentrations of IL-22 correlated with those of IL-17A (r = 0.726; P < 0.001). These results suggest that both IL-17A and IL-22 affect development of CCA related to liver fluke infection. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

The morphology and ultrastructure of salivary glands of Zoraptera (Insecta).

PubMed

Dallai, R; Mercati, D; Mashimo, Y; Machida, R; Beutel, R G

2017-07-01

The salivary glands of two species of Zoraptera, Zorotypus caudelli and Zorotypus hubbardi, were examined and documented mainly using transmission electron microscopy (TEM). The results obtained for males and females of the two species are compared and functional aspects related to ultrastructural features are discussed. The salivary glands are divided into two regions: the secretory cell region and the long efferent duct, the latter with its distal end opening in the salivarium below the hypopharyngeal base. The secretory region consists of a complex of secretory cells provided with microvillated cavities connected by short ectodermal ducts to large ones, which are connected with the long efferent duct. The secretory cell cytoplasm contains a large system of rough endoplasmic reticulum and Golgi apparatus producing numerous dense secretions. The cells of the efferent duct, characterized by reduced cytoplasm and the presence of long membrane infoldings associated with mitochondria, are possibly involved in fluid uptaking from the duct lumen. Copyright © 2017 Elsevier Ltd. All rights reserved.

High energy density aluminum-oxygen cell

NASA Technical Reports Server (NTRS)

Rudd, E. J.; Gibbons, D. W.

1993-01-01

An alternative to a secondary battery as the power source for vehicle propulsion is a fuel cell. An example of this is the metal-air fuel cell using metals such as aluminum, zinc, or iron. Aluminum is a particularly attractive candidate, having high energy and power densities, being environmentally acceptable, and having a large, established industrial base for production and distribution. An aluminum-oxygen system is currently under development for a UUV test vehicle, and recent work has focussed upon low corrosion aluminum alloys and an electrolyte management system for processing the by-products of the energy-producing reactions. This paper summarizes the progress made in both areas. Anode materials capable of providing high utilization factors over current densities ranging from S to 150 mA/sq cm have been identified. These materials are essential to realizing an acceptable mission life for the UUV. With respect to the electrolyte management system, a filter/precipitator unit has been successfully operated for over 250 hours in a large scale, half-cell system.

Vaccine manufacturing and technology: from biotechnological platforms to syntethic epitopes, current viepoint.

PubMed

Ignateva, G A

2016-01-01

The Purposes: the review take into account short history of vaccination practice and development of vaccine technology. In the review we include data from several monographs about manufacturing of vaccines published by authors from such companies as Merck & Co; Sanofi Pasteur; Dynavax Europe/Rhein Biotech GmbH; Latham Biopharm Group; Aridis Pharmaceuticals LLC; Genentech; Amgen; Shamir Biologics LLC; Biopharm Services US; Novartis Pharma AG, аnd several research centers: Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research; Purdue University, West Lafayette, IN, US; Department of Pharmaceutical Chemistry, Univ. Of Kansas; Max Planck Institute for dynamics of Complex Technical Systems; Fraunhofer USA Center for Molecular Biotechnology; US Dep. of Agriculture Animal and Plant Health Inspection Service, etc. In historic literature there are data about inoculation practices in antique China, Persia, India, Byzantium, native Americans, some African population. In modern immunology since the end of XIX century the vaccines were produced at the in vivo platforms - in animals (rabbits, mice, cows). Since 1931 due to E. Goodpasture' elaboration most virus vaccines were and are produced at the in ovo platform. In 1949 J.F. Enders elaborated large-scale polio virus production in the primary culture of monkey kidney cells in vitro. Up to day primary culture of chiken embrio fibroblasts are used to large-scale production of vaccine viruses of measles, mumps, rabies. Since 2000-th in Western countries most part of virus vaccines were began to produced via a cultivation in continuous tumor cell lines. The last technology is the most low cost for large-scale production of vaccines. We review several new biotechnological platforms for the production of the recombinant protein or virus-like particles as subunit vaccines: plant system, algae, mushrooms, insect cells, etc. Beside of good purpose of vaccination - prophylactic of several infectious deseases, doctors must take into account possibility of inter-species transmission of unknown pathogens (retroviruses, prions, etc) from biotechnological platforms - animals, cell cultures - into human population, and don't ignore L.A. Zilber' theory of virus' etiology of cancer diseases.

Transient removal of alkaline zones after excitation of Chara cells is associated with inactivation of high conductance in the plasmalemma

PubMed Central

2009-01-01

The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15–30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H+ influx (OH− efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H+/HCO3− symport or OH−/HCO3− antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions. PMID:19820298

Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche.

PubMed

Baghdadi, Meryem B; Castel, David; Machado, Léo; Fukada, So-Ichiro; Birk, David E; Relaix, Frederic; Tajbakhsh, Shahragim; Mourikis, Philippos

2018-05-01

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix 1-3 . Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells 4-9 , the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.

GCR Transport in the Brain: Assessment of Self-Shielding, Columnar Damage, and Nuclear Reactions on Cell Inactivation Rates

NASA Technical Reports Server (NTRS)

Shavers, M. R.; Atwell, W.; Cucinotta, F. A.; Badhwar, G. D. (Technical Monitor)

1999-01-01

Radiation shield design is driven by the need to limit radiation risks while optimizing risk reduction with launch mass/expense penalties. Both limitation and optimization objectives require the development of accurate and complete means for evaluating the effectiveness of various shield materials and body-self shielding. For galactic cosmic rays (GCR), biophysical response models indicate that track structure effects lead to substantially different assessments of shielding effectiveness relative to assessments based on LET-dependent quality factors. Methods for assessing risk to the central nervous system (CNS) from heavy ions are poorly understood at this time. High-energy and charge (HZE) ion can produce tissue events resulting in damage to clusters of cells in a columnar fashion, especially for stopping heavy ions. Grahn (1973) and Todd (1986) have discussed a microlesion concept or model of stochastic tissue events in analyzing damage from HZE's. Some tissues, including the CNS, maybe sensitive to microlesion's or stochastic tissue events in a manner not illuminated by either conventional dosimetry or fluence-based risk factors. HZE ions may also produce important lateral damage to adjacent cells. Fluences of high-energy proton and alpha particles in the GCR are many times higher than HZE ions. Behind spacecraft and body self-shielding the ratio of protons, alpha particles, and neutrons to HZE ions increases several-fold from free-space values. Models of GCR damage behind shielding have placed large concern on the role of target fragments produced from tissue atoms. The self-shielding of the brain reduces the number of heavy ions reaching the interior regions by a large amount and the remaining light particle environment (protons, neutrons, deuterons. and alpha particles) may be the greatest concern. Tracks of high-energy proton produce nuclear reactions in tissue, which can deposit doses of more than 1 Gv within 5 - 10 cell layers. Information on rates of cell killing from GCR, including patterns of cell killing from single particle tracks. can provide useful information on expected differences between proton and HZE tracks and clinical experiences with photon irradiation. To model effects on cells in the brain, it is important that transport models accurately describe changes in the GCR due to interactions in the cranium and proximate tissues. We describe calculations of the attenuated GCR particle fluxes at three dose-points in the brain and associated patterns of cell killing using biophysical models. The effects of the brain self-shielding and bone-tissue interface of the skull in modulating the GCR environment are considered. For each brain dose-point, the mass distribution in the surrounding 4(pi) solid angle is characterized using the CAM model to trace 512 rays. The CAM model describes the self-shielding by converting the tissue distribution to mass-equivalent aluminum, and nominal values of spacecraft shielding is considered. Particle transport is performed with the proton, neutron, and heavy-ion transport code HZETRN with the nuclear fragmentation model QMSFRG. The distribution of cells killed along the path of individual GCR ions is modeled using in vitro cell inactivation data for cells with varying sensitivity. Monte Carlo simulations of arrays of inactivated cells are considered for protons and heavy ions and used to describe the absolute number of cell killing events of various magnitude in the brain from the GCR. Included are simulations of positions of inactivated cells from stopping heavy ions and nuclear stars produced by high-energy ions most importantly, protons and neutrons.

Production of human monoclonal antibody in eggs of chimeric chickens.

PubMed

Zhu, Lei; van de Lavoir, Marie-Cecile; Albanese, Jenny; Beenhouwer, David O; Cardarelli, Pina M; Cuison, Severino; Deng, David F; Deshpande, Shrikant; Diamond, Jennifer H; Green, Lynae; Halk, Edward L; Heyer, Babette S; Kay, Robert M; Kerchner, Allyn; Leighton, Philip A; Mather, Christine M; Morrison, Sherie L; Nikolov, Zivko L; Passmore, David B; Pradas-Monne, Alicia; Preston, Benjamin T; Rangan, Vangipuram S; Shi, Mingxia; Srinivasan, Mohan; White, Steven G; Winters-Digiacinto, Peggy; Wong, Susan; Zhou, Wen; Etches, Robert J

2005-09-01

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

Recent progress in terrestrial photovoltaic collector technology

NASA Technical Reports Server (NTRS)

Ferber, R. R.

1982-01-01

The U.S. Photovoltaic Research and Development Program has the objective to develop the technology necessary to foster widespread grid-competitive electric power generation by the late 1980s. The flat-plate and the concentrator collector activities form the nucleus of the program. The project is concerned with the refining of silicon, silicon sheet production, solar cell processing and fabrication, encapsulation materials development, and collector design and production. The Large-Area Silicon Sheet Task has the objective to develop and demonstrate the feasibility of several methods for producing large area silicon sheet material suitable for fabricating low-cost, high-efficiency solar cells. It is expected that a variety of economic flat-plate and concentrator collectors will become commercially available for grid-connected applications.

Characterization of a biosurfactant produced by Pseudomonas cepacia CCT6659 in the presence of industrial wastes and its application in the biodegradation of hydrophobic compounds in soil.

PubMed

Silva, Elias J; Rocha e Silva, Nathália Maria P; Rufino, Raquel D; Luna, Juliana M; Silva, Ricardo O; Sarubbo, Leonie A

2014-05-01

The bacterium Pseudomonas cepacia CCT6659 cultivated with 2% soybean waste frying oil and 2% corn steep liquor as substrates produced a biosurfactant with potential application in the bioremediation of soils. The biosurfactant was classified as an anionic biomolecule composed of 75% lipids and 25% carbohydrates. Characterization by proton nuclear magnetic resonance ((1)H and (13)C NMR) revealed the presence of carbonyl, olefinic and aliphatic groups, with typical spectra of lipids. Four sets of biodegradation experiments were carried out with soil contaminated by hydrophobic organic compounds amended with molasses in the presence of an indigenous consortium, as follows: Set 1-soil+bacterial cells; Set 2-soil+biosurfactant; Set 3-soil+bacterial cells+biosurfactant; and Set 4-soil without bacterial cells or biosurfactant (control). Significant oil biodegradation activity (83%) occurred in the first 10 days of the experiments when the biosurfactant and bacterial cells were used together (Set 3), while maximum degradation of the organic compounds (above 95%) was found in Sets 1-3 between 35 and 60 days. It is evident from the results that the biosurfactant alone and its producer species are both capable of promoting biodegradation to a large extent. Copyright © 2014 Elsevier B.V. All rights reserved.

The effect of single large dose hydrocortisone treatment on IgM and IgG antibody production, morphological distribution of antibody producing cells and immunological memory

PubMed Central

Petrányi, Gy.; Benczúr, M.; Alföldy, P.

1971-01-01

The effect of a single subcutaneous dose of hydrocortisone (730 mg/kg ∼ 21-day LD50) on the haemolysin response of mice to sheep erythrocyte antigen was examined. Hydrocortisone was administered once at times varying from 5 days before immunization with sheep erythrocytes to 9 days after antigen. Total suppression of the 7S haemolysin titre was brought about by treatment with single dose of 750 mg/kg in the period 5 days prior to antigen until 2 days after antigen; at the same time, the titre of 19S haemolysin exceeded the control 19S titre. Microplaque assay on the 5th day failed to confirm total suppression of 7S antibody synthesis, as 4 per cent of the splenic plaque-forming cells produced 7S. The same assay failed to verify the augmentation of 19S production on a cellular level, as the number of 19S plaque-forming was significantly decreased. Hydrocortisone could be shown to influence the morphology of the 19S antibody producing cells by increasing the percentage of mature cell types. A selective depressing activity by hydrocortisone on 7S memory was found. The theoretical implications of these findings are discussed. PMID:4934137

Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion.

PubMed

Yan, Jing; Nadell, Carey D; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

2017-08-23

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species.Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line.

PubMed

Pushparajan, Charlotte; Claus, Juan Daniel; Marshall, Sean David Goldie; Visnovsky, Gabriel

2013-12-01

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctes rhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchorage-dependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express™ with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 × 10(5) cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h(-1)). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 × 10(7) TCID50/ml) than in cultures infected in Sf-900 III (2.0 × 10(7) TCID50/ml) and Sf-900 II (1.4 × 10(7) TCID50/ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.

Automated array assembly task development of low-cost polysilicon solar cells

NASA Technical Reports Server (NTRS)

Jones, G. T.

1980-01-01

Development of low cost, large area polysilicon solar cells was conducted in this program. Three types of polysilicon materialk were investigated. A theoretical and experimenal comparison between single crystal silicon and polysilicon solar cell efficiency was performed. Significant electrical performance differences were observed between types of wafer material, i.e. fine grain and coarse grain polysilicon and single crystal silicon. Efficiency degradation due to grain boundaries in fin grain and coarse grain polysilicon was shown to be small. It was demonstrated that 10 percent efficient polysilicon solar cells can be produced with spray on n+ dopants. This result fulfills an important goal of this project, which is the production of batch quantity of 10 percent efficient polysilicon solar cells.

Rectification of Acetylcholine-Elicited Currents in PC12 Pheochromocytoma Cells

NASA Astrophysics Data System (ADS)

Ifune, C. K.; Steinbach, J. H.

1990-06-01

The current-voltage (I-V) relationship for acetylcholine-elicited currents in the rat pheochromocytoma cell line PC12 is nonlinear. Two voltage-dependent processes that could account for the whole-cell current rectification were examined, receptor channel gating and single receptor channel permeation. We found that both factors are involved in the rectification of the whole-cell currents. The voltage dependence of channel gating determines the shape of the I-V curve at negative potentials. The single-channel I-V relationship is inwardly rectifying and largely responsible for the characteristic shape of the whole-cell I-V curve at positive potentials. The rectification of the single-channel currents is produced by the voltage-dependent block of outward currents by intracellular Mg2+ ions.

Gram-scale synthesis of single-crystalline graphene quantum dots with superior optical properties.

PubMed

Wang, Liang; Wang, Yanli; Xu, Tao; Liao, Haobo; Yao, Chenjie; Liu, Yuan; Li, Zhen; Chen, Zhiwen; Pan, Dengyu; Sun, Litao; Wu, Minghong

2014-10-28

Graphene quantum dots (GQDs) have various alluring properties and potential applications, but their large-scale applications are limited by current synthetic methods that commonly produce GQDs in small amounts. Moreover, GQDs usually exhibit polycrystalline or highly defective structures and thus poor optical properties. Here we report the gram-scale synthesis of single-crystalline GQDs by a facile molecular fusion route under mild and green hydrothermal conditions. The synthesis involves the nitration of pyrene followed by hydrothermal treatment in alkaline aqueous solutions, where alkaline species play a crucial role in tuning their size, functionalization and optical properties. The single-crystalline GQDs are bestowed with excellent optical properties such as bright excitonic fluorescence, strong excitonic absorption bands extending to the visible region, large molar extinction coefficients and long-term photostability. These high-quality GQDs can find a large array of novel applications in bioimaging, biosensing, light emitting diodes, solar cells, hydrogen production, fuel cells and supercapacitors.

Repair of large full-thickness articular cartilage defects in the rabbit: the effects of joint distraction and autologous bone-marrow-derived mesenchymal cell transplantation.

PubMed

Yanai, T; Ishii, T; Chang, F; Ochiai, N

2005-05-01

We produced large full-thickness articular cartilage defects in 33 rabbits in order to evaluate the effect of joint distraction and autologous culture-expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT) at 12 weeks. After fixing the knee on a hinged external fixator, we resected the entire surface of the tibial plateau. We studied three groups: 1) with and without joint distraction; 2) with joint distraction and collagen gel, and 3) with joint distraction and ACBMT and collagen gel. The histological scores were significantly higher in the groups with ACBMT collagen gel (p < 0.05). The area of regenerated soft tissue was smaller in the group allowed to bear weight (p < 0.05). These findings suggest that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel and ACBMT.

Electromagnetic needles with submicron pole tip radii for nanomanipulation of biomolecules and living cells

NASA Astrophysics Data System (ADS)

Matthews, Benjamin D.; LaVan, David A.; Overby, Darryl R.; Karavitis, John; Ingber, Donald E.

2004-10-01

We describe the design and fabrication of a temperature-controlled electromagnetic microneedle (EMN) to generate custom magnetic field gradients for biomedical and biophysical applications. An electropolishing technique was developed to sharpen the EMN pole tip to any desired radius between 100 nm and 20 μm. The EMN can be used to apply strong static or dynamic forces (>50nN) to micrometer- or nanometer-sized magnetic beads without producing significant heating or needle movement. Large tip radii (20 μm) allow magnetic force application to multiple magnetic beads over a large area, while small radii (0.1-6 μm) can be used to selectively pull or capture single magnetic beads from within a large population of similar particles. The customizable EMN is thus well suited for micro- and nanomanipulation of magnetic particles linked to biomolecules or living cells.

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

PubMed Central

Tornow, J; Polvino-Bodnar, M; Santangelo, G; Cole, C N

1985-01-01

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain. Images PMID:2982029

Fabrication, vascularization and osteogenic properties of a novel synthetic biomimetic induced membrane for the treatment of large bone defects

PubMed Central

Browne, Christopher; Bishop, Julius; Yang, Yunzhi

2014-01-01

The induced membrane has been widely used in the treatment of large bone defects but continues to be limited by a relatively lengthy healing process and a requisite two stage surgical procedure. Here we report the development and characterization of a synthetic biomimetic induced membrane (BIM) consisting of an inner highly pre-vascularized cell sheet and an outer osteogenic layer using cell sheet engineering. The pre-vascularized inner layer was formed by seeding human umbilical vein endothelial cells (HUVECs) on a cell sheet comprised of a layer of undifferentiated human bone marrow-derived mesenchymal stem cells (hMSCs). The outer osteogenic layer was formed by inducing osteogenic differentiation of hMSCs. In vitro results indicated the undifferentiated hMSCs cell sheet facilitated the alignment of HUVECs and significantly promoted the formation of vascular-like networks. Furthermore, seeded HUVECs rearranged the extracellular matrix produced by hMSCs sheet. After subcutaneously implantation, the composite constructs showed rapid vascularization and anastomosis with the host vascular system, forming functional blood vessels in vivo. Osteogenic potential of the BIM was evidenced by immunohistochemistry staining of osteocalcin, tartrate-resistant acid phosphatase (TRAP) staining, and alizarin red staining. In summary, the synthetic BIM showed rapid vascularization, significant anastomoses, and osteogenic potential in vivo. This synthetic BIM has the potential for treatment of large bone defects in the absence of infection. PMID:24747351

Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and β-glucosidase.

PubMed

Apiwatanapiwat, Waraporn; Murata, Yoshinori; Kosugi, Akihiko; Yamada, Ryosuke; Kondo, Akihiko; Arai, Takamitsu; Rugthaworn, Prapassorn; Mori, Yutaka

2011-04-01

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

Cytotoxic Th1 and Th17 cells infiltrate the intestinal mucosa of Behcet patients and exhibit high levels of TNF-α in early phases of the disease

PubMed Central

Emmi, Giacomo; Silvestri, Elena; Bella, Chiara Della; Grassi, Alessia; Benagiano, Marisa; Cianchi, Fabio; Squatrito, Danilo; Cantarini, Luca; Emmi, Lorenzo; Selmi, Carlo; Prisco, Domenico; D’Elios, Mario Milco

2016-01-01

Abstract Background: Gastrointestinal involvement is one of the most serious in Behçet disease, potentially leading to severe complications. Aim of this study was to investigate at mucosal level the T-cell responses in Behçet patients with early intestinal involvement. Methods: We isolated T cells from intestinal mucosa of 8 patients with intestinal symptoms started within 6 months. T lymphocytes were cloned and analyzed for surface phenotype and cytokines production. Results: We obtained 382 T-cell clones: 324 were CD4+ and 58 were CD8+. Within the 324 CD4+ clones, 195 were able to secrete IFN-γ and TNF-α, but not IL-4, nor IL-17 thus showing a polarized Th1 profile, whereas CD4 clones producing both IFN-γ and IL-17 (Th1/Th17 profile) were 79. Likewise, the number of CD8 clones producing type 1 cytokines was higher than those of CD8 clones producing both type 1 and 2 cytokines. Almost all intestinal-derived T-cell clones expressed perforin-mediated cytotoxicity and Fas–Fas Ligand-mediated pro-apoptotic activity. Conclusions: Our results indicate that in the early stages of the disease, both Th1 and Th17 cells drive inflammation leading to mucosal damage via abnormal and long-lasting cytokines production as well as via both perforin- and Fas–Fas ligand-mediated cytotoxicity. Finally, all the T cells at mucosal level were able to produce large amount of TNF-α, suggesting that its production is a property of intestinal T cells of patients with early active intestinal disease. These results support the therapy with anti-TNF-α agents and suggest the use of anti-IL-17 monoclonal antibodies in Behçet patients with early intestinal involvement. PMID:27930541

Bone cells in birds show exceptional surface area, a characteristic tracing back to saurischian dinosaurs of the late Triassic.

PubMed

Rensberger, John M; Martínez, Ricardo N

2015-01-01

Dinosaurs are unique among terrestrial tetrapods in their body sizes, which range from less than 3 gm in hummingbirds to 70,000 kg or more in sauropods. Studies of the microstructure of bone tissue have indicated that large dinosaurs, once believed to be slow growing, attained maturity at rates comparable to or greater than those of large mammals. A number of structural criteria in bone tissue have been used to assess differences in rates of osteogenesis in extinct taxa, including counts of lines of arrested growth and the density of vascular canals. Here, we examine the density of the cytoplasmic surface of bone-producing cells, a feature which may set an upper limit to the rate of osteogenesis. Osteocyte lacunae and canaliculi, the cavities in bone containing osteocytes and their extensions, were measured in thin-sections of primary (woven and parallel fibered) bone in a diversity of tetrapods. The results indicate that bone cell surfaces are more densely organized in the Saurischia (extant birds, extinct Mesozoic Theropoda and Sauropodomorpha) than in other tetrapods, a result of denser branching of the cell extensions. The highest postnatal growth rates among extant tetrapods occur in modern birds, the only surviving saurischians, and the finding of exceptional cytoplasmic surface area of the cells that produce bone in this group suggests a relationship with bone growth rate. In support of this relationship is finding the lowest cell surface density among the saurischians examined in Dinornis, a member of a group of ratites that evolved in New Zealand in isolation from mammalian predators and show other evidence of lowered maturation rates.

Bone Cells in Birds Show Exceptional Surface Area, a Characteristic Tracing Back to Saurischian Dinosaurs of the Late Triassic

PubMed Central

Rensberger, John M.; Martínez, Ricardo N.

2015-01-01

Background Dinosaurs are unique among terrestrial tetrapods in their body sizes, which range from less than 3 gm in hummingbirds to 70,000 kg or more in sauropods. Studies of the microstructure of bone tissue have indicated that large dinosaurs, once believed to be slow growing, attained maturity at rates comparable to or greater than those of large mammals. A number of structural criteria in bone tissue have been used to assess differences in rates of osteogenesis in extinct taxa, including counts of lines of arrested growth and the density of vascular canals. Methodology/Principal Findings Here, we examine the density of the cytoplasmic surface of bone-producing cells, a feature which may set an upper limit to the rate of osteogenesis. Osteocyte lacunae and canaliculi, the cavities in bone containing osteocytes and their extensions, were measured in thin-sections of primary (woven and parallel fibered) bone in a diversity of tetrapods. The results indicate that bone cell surfaces are more densely organized in the Saurischia (extant birds, extinct Mesozoic Theropoda and Sauropodomorpha) than in other tetrapods, a result of denser branching of the cell extensions. The highest postnatal growth rates among extant tetrapods occur in modern birds, the only surviving saurischians, and the finding of exceptional cytoplasmic surface area of the cells that produce bone in this group suggests a relationship with bone growth rate. In support of this relationship is finding the lowest cell surface density among the saurischians examined in Dinornis, a member of a group of ratites that evolved in New Zealand in isolation from mammalian predators and show other evidence of lowered maturation rates. PMID:25830561

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

PubMed Central

Herrera, Elizabeth; del Mar Lorenzo, María; Blasco, Rafael; Isaacs, Stuart N.

1998-01-01

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses. PMID:9420227

Can mesenchymal cells undergo collective cell migration?

PubMed Central

Theveneau, Eric

2011-01-01

Cell migration is critical for proper development of the embryo and is also used by many cell types to perform their physiological function. For instance, cell migration is essential for immune cells to monitor the body and for epithelial cells to heal a wound whereas, in cancer cells, acquisition of migratory capabilities is a critical step toward malignancy. Migratory cells are often categorized into two groups: (1) mesenchymal cells, produced by an epithelium-to-mesenchyme transition, that undergo solitary migration and (2) epithelial-like cells which migrate collectively. However, on some occasions, mesenchymal cells may travel in large, dense groups and exhibit key features of collectively migrating cells such as coordination and cooperation. Here, using data published on neural crest cells, a highly invasive mesenchymal cell population that extensively migrate throughout the embryo, we explore the idea that mesenchymal cells, including cancer cells, might be able to undergo collective cell migration under certain conditions and discuss how they could do so. PMID:22274714

High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors.

PubMed

Koizume, Shiro; Ito, Shin; Yoshioka, Yusuke; Kanayama, Tomohiko; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Yamada, Roppei; Ochiya, Takahiro; Ruf, Wolfram; Miyagi, Etsuko; Hirahara, Fumiki; Miyagi, Yohei

2016-01-01

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

Use of biochemical lesions for selection of human cells with hybrid cytoplasms.

PubMed Central

Wright, W E; Hayflick, L

1975-01-01

Techniques for preparing large populations of anucleate cytoplasms from cultured eukaryotic cells have only recently been described. The principal value of anucleate cytoplasms derives from studies that can be done after they are fused to whole cells. Since present methods for the isolation of heterokaryons are unsuitable for the selection of hybrids between whole cells and anucleate cytoplasms (heteroplasmons), a selective system has been developed which is based on the capacity of anucleate cytoplasms containing active enzymes to rescue whole cells poisoned with iodoacetate. Ethidium bromide, a partially effective agent, was used in conjunction with iodoacetate to demonstrate the feasibility of selecting heterokaryons by producing complementary biochemical lesions in the parental cell strains. The potential for artifact in these systems is not, however, entirely precluded. Images PMID:1057172

Fixation of carbon dioxide by a hydrogen-oxidizing bacterium for value-added products.

PubMed

Yu, Jian

2018-06-09

With rapid technology progress and cost reduction, clean hydrogen from water electrolysis driven by renewable powers becomes a potential feedstock for CO 2 fixation by hydrogen-oxidizing bacteria. Cupriavidus necator (formally Ralstonia eutropha), a representative member of the lithoautotrophic prokaryotes, is a promising producer of polyhydroxyalkanoates and single cell proteins. This paper reviews the fundamental properties of the hydrogen-oxidizing bacterium, the metabolic activities under limitation of individual gases and nutrients, and the value-added products from CO 2 , including the products with large potential markets. Gas fermentation and bioreactor safety are discussed for achieving high cell density and high productivity of desired products under chemolithotrophic conditions. The review also updates the recent research activities in metabolic engineering of C. necator to produce novel metabolites from CO 2 .

Thermodynamics and Transport Phenomena in High Temperature Steam Electrolysis Cells

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

James E. O'Brien

2012-03-01

Hydrogen can be produced from water splitting with relatively high efficiency using high temperature electrolysis. This technology makes use of solid-oxide cells, running in the electrolysis mode to produce hydrogen from steam, while consuming electricity and high temperature process heat. The overall thermal-to-hydrogen efficiency for high temperature electrolysis can be as high as 50%, which is about double the overall efficiency of conventional low-temperature electrolysis. Current large-scale hydrogen production is based almost exclusively on steam reforming of methane, a method that consumes a precious fossil fuel while emitting carbon dioxide to the atmosphere. An overview of high temperature electrolysis technologymore » will be presented, including basic thermodynamics, experimental methods, heat and mass transfer phenomena, and computational fluid dynamics modeling.« less

Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals.

PubMed

Langseth, W; Bernhoft, A; Rundberget, T; Kosiak, B; Gareis, M

Thirty-four isolates of the eight most common Fusarium species isolated from Norwegian cereals; F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. sporotrichioides, F. torulosum and F. tricinctum were studied for their cytotoxicity and ability to produce mycotoxins. The strains were cultivated on rice, and analysed for trichothecenes (all species), zearalenone (all species), fusarochromanone (F. equiseti), wortmannin (F. torulosum), moniliformin and enniatins (F. avenaceum, F. tricinctum and F. torulosum). The cytotoxicity of the extracts were examined with an (in vitro) MTT-cell culture assay. All F. graminearum and five of seven F. culmorum isolates belonged to chemotype IA, producing deoxynivalenol and 3-acetyl-deoxynivalenol, while the two other F. culmorum strains were nivalenol producers (chemotype II). The F. equiseti isolates and one of the F. poae isolates produced both type A and B trichothecenes, and relatively large quantities of fusarochromanone were detected in the F. equiseti cultures. All Fusarium species studied showed significant cytotoxicity, but with a large variation between species, and also within each species. F. sporotrichioides and F. equiseti showed the highest average cytotoxicity.

Wide-band tracheids are present in almost all species of Cactaceae.

PubMed

Mauseth, James D

2004-02-01

Wide-band tracheids (WBTs) have been found in seedlings of most species of cacti that have fibrous wood in their adult bodies. Consequently, this cell type is now known to be present in almost all cacti. Earlier studies of adult plants revealed WBTs to be present only in cacti with globose or short, broad bodies, whereas all species with large columnar or long slender bodies had fibrous wood without WBTs. However, even these species produce WBTs during the first several months after germination. In species with fibrous wood in their adult bodies (species with large or slender bodies), seedlings undergo a phase transition in wood morphogenesis after a few months and stop producing the juvenile (WBT) wood and begin producing adult (fibrous) wood. If adult plants have an intermediate size, the phase transition is delayed and the plant produces WBT wood for several years. Species with globose bodies repress the phase transition completely and never switch to producing adult (fibrous) wood. Because WBTs are so widespread, they probably originated only once in Cactaceae, not multiple times as suggested earlier, or there may have been just a single origin in the Cactaceae/Portulacaceae clade.

Molecular characterization of baculovirus Bombyx mori nucleopolyhedrovirus polyhedron mutants.

PubMed

Katsuma, S; Noguchi, Y; Shimada, T; Nagata, M; Kobayashi, M; Maeda, S

1999-01-01

Four newly isolated and two previously isolated polyhedron mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Two polyhedron deficient mutants, #126 and #136, produced small uncrystallized particles of polyhedrin in the nuclei and cytoplasm of infected cells. Mutant #211 produced a large number of variably sized polyhedra in the nucleus and #220 produced a few large cuboidal polyhedra in the nucleus. Mutant #24 and #128 were previously isolated BmNPV mutants. Mutant #24 could not produce polyhedrin mRNA and polyhedra produced by mutant #128 lacked oral infectivity. Nucleotide sequence analysis indicated that five mutants (#126, #136, #211, #220 and #128) had amino acid substitutions in polyhedrin and mutant #24 had a point mutation only in the promoter region of the polyhedrin gene. Cotransfection experiments showed that the altered phenotypes were due to the mutations found in the polyhedrin gene regions. In mutants #126 and #136, amino acid sequences of the nuclear localization signal of polyhedrin were identical to those of wild-type BmNPV, suggesting that this sequence was necessary but not sufficient for nuclear localization of polyhedrin. Electron microscopic observation revealed that fewer occluded virions were contained in polyhedra of #128 and #220.

Demonstration-Scale High-Cell-Density Fermentation of Pichia pastoris.

PubMed

Liu, Wan-Cang; Zhu, Ping

2018-01-01

Pichia pastoris has been one of the most successful heterologous overexpression systems in generating proteins for large-scale production through high-cell-density fermentation. However, optimizing conditions of the large-scale high-cell-density fermentation for biochemistry and industrialization is usually a laborious and time-consuming process. Furthermore, it is often difficult to produce authentic proteins in large quantities, which is a major obstacle for functional and structural features analysis and industrial application. For these reasons, we have developed a protocol for efficient demonstration-scale high-cell-density fermentation of P. pastoris, which employs a new methanol-feeding strategy-biomass-stat strategy and a strategy of increased air pressure instead of pure oxygen supplement. The protocol included three typical stages of glycerol batch fermentation (initial culture phase), glycerol fed-batch fermentation (biomass accumulation phase), and methanol fed-batch fermentation (induction phase), which allows direct online-monitoring of fermentation conditions, including broth pH, temperature, DO, anti-foam generation, and feeding of glycerol and methanol. Using this protocol, production of the recombinant β-xylosidase of Lentinula edodes origin in 1000-L scale fermentation can be up to ~900 mg/L or 9.4 mg/g cells (dry cell weight, intracellular expression), with the specific production rate and average specific production of 0.1 mg/g/h and 0.081 mg/g/h, respectively. The methodology described in this protocol can be easily transferred to other systems, and eligible to scale up for a large number of proteins used in either the scientific studies or commercial purposes.

Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

PubMed Central

Fu, X Y; Colgan, J D; Manley, J L

1988-01-01

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells. Images PMID:2851720

Redundant role of tissue-selective TAF(II)105 in B lymphocytes.

PubMed

Freiman, Richard N; Albright, Shane R; Chu, Leslie E; Zheng, Shuang; Liang, Hong-Erh; Sha, William C; Tjian, Robert

2002-09-01

Regulated gene expression is a complex process achieved through the function of multiple protein factors acting in concert at a given promoter. The transcription factor TFIID is a central component of the machinery regulating mRNA synthesis by RNA polymerase II. This large multiprotein complex is composed of the TATA box binding protein (TBP) and several TBP-associated factors (TAF(II)s). The recent discovery of multiple TBP-related factors and tissue-specific TAF(II)s suggests the existence of specialized TFIID complexes that likely play a critical role in regulating transcription in a gene- and tissue-specific manner. The tissue-selective factor TAF(II)105 was originally identified as a component of TFIID derived from a human B-cell line. In this report we demonstrate the specific induction of TAF(II)105 in cultured B cells in response to bacterial lipopolysaccharide (LPS). To examine the in vivo role of TAF(II)105, we have generated TAF(II)105-null mice by homologous recombination. Here we show that B-lymphocyte development is largely unaffected by the absence of TAF(II)105. TAF(II)105-null B cells can proliferate in response to LPS, produce relatively normal levels of resting antibodies, and can mount an immune response by producing antigen-specific antibodies in response to immunization. Taken together, we conclude that the function of TAF(II)105 in B cells is likely redundant with the function of other TAF(II)105-related cellular proteins.

A Simple and Rapid Protocol for Producing Yeast Extract from Saccharomyces cerevisiae Suitable for Preparing Bacterial Culture Media

PubMed Central

Zarei, Omid; Dastmalchi, Siavoush; Hamzeh-Mivehroud, Maryam

2016-01-01

Yeasts, especially Saccharomyces cerevisiae, are one of the oldest organisms with broad spectrum of applications, owing to their unique genetics and physiology. Yeast extract, i.e. the product of yeast cells, is extensively used as nutritional resource in bacterial culture media. The aim of this study was to develop a simple, rapid and cost benefit process to produce the yeast extract. In this procedure mechanical methods such as high temperature and pressure were utilized to produce the yeast extract. The growth of the bacteria feed with the produced yeast extract was monitored in order to assess the quality of the product. The results showed that the quality of the produced yeast extract was very promising concluded from the growth pattern of bacterial cells in media prepared from this product and was comparable with that of the three commercial yeast extracts in terms of bacterial growth properties. One of the main advantages of the current method was that no chemicals and enzymes were used, leading to the reduced production cost. The method is very simple and cost effective, and can be performed in a reasonable time making it suitable for being adopted by research laboratories. Furthermore, it can be scaled up to produce large quantities for industrial applications. PMID:28243289

Noncoordinate expression of J-chain and Blimp-1 define nurse shark plasma cell populations during ontogeny.

PubMed

Castro, Caitlin D; Ohta, Yuko; Dooley, Helen; Flajnik, Martin F

2013-11-01

B-lymphocyte-induced maturation protein 1 (Blimp-1) is the master regulator of plasma cell development, controlling genes such as those encoding J-chain and secretory Ig heavy chain. However, some mammalian plasma cells do not express J-chain, and mammalian B1 cells secrete "natural" IgM antibodies without upregulating Blimp-1. While these results have been controversial in mammalian systems, here we describe subsets of normally occurring Blimp-1(-) antibody-secreting cells in nurse sharks, found in lymphoid tissues at all ontogenic stages. Sharks naturally produce large amounts of both pentameric (classically "19S") and monomeric (classically "7S") IgM, the latter an indicator of adaptive immunity. Consistent with the mammalian paradigm, shark Blimp-1 is expressed in splenic 7S IgM-secreting cells, though rarely detected in the J-chain(+) cells producing 19S IgM. Although IgM transcript levels are lower in J-chain(+) cells, these cells nevertheless secrete 19S IgM in the absence of Blimp-1, as demonstrated by ELISPOT and metabolic labeling. Additionally, cells in the shark BM equivalent (epigonal) are Blimp-1(-). Our data suggest that, in sharks, 19S-secreting cells and other secreting memory B cells in the epigonal are maintained for long periods without Blimp-1, but like in mammals, Blimp-1 is required for terminating the B-cell program following an adaptive immune response in the spleen. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-coordinate expression of J-chain and Blimp-1 define nurse shark plasma cell populations during ontogeny

PubMed Central

Castro, Caitlin D.; Ohta, Yuko; Dooley, Helen; Flajnik, Martin F.

2014-01-01

Summary Blimp-1 is the master regulator of plasma cell development, controlling genes such as J-chain and secretory Ig heavy chain. However, some mammalian plasma cells do not express J-chain, and mammalian B1 cells secrete “natural” IgM antibodies without upregulating Blimp-1. While these results have been controversial in mammalian systems, here we describe subsets of normally occurring Blimp-1- antibody secreting cells in nurse sharks, found in lymphoid tissues at all ontogenic stages. Sharks naturally produce large amounts of both pentameric (classically ‘19S’) and monomeric (classically ‘7S’) IgM, the latter an indicator of adaptive immunity. Consistent with the mammalian paradigm, shark Blimp-1 is expressed in splenic 7S IgM-secreting cells, though rarely detected in the J-chain+ cells producing 19S IgM. Although IgM transcript levels are lower in J-chain+ cells, these cells nevertheless secrete 19S IgM in the absence of Blimp-1, as demonstrated by ELISPOT and metabolic labeling. Additionally, cells in the shark bone marrow equivalent (epigonal) are Blimp-1-. Our data suggest that, in sharks, 19S-secreting cells and other secreting memory B cells in the epigonal can be maintained for long periods without Blimp-1, but like in mammals, Blimp-1 is required for terminating the B cell program following an adaptive immune response in the spleen. PMID:23897025

Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

PubMed Central

Uhlemann, Eva-Maria E; Pierson, Hannah E; Fillingame, Robert H; Dmitriev, Oleg Y

2012-01-01

NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes. PMID:22162071

Differentiation and Behavior of Dental Pulp Stem Cells in Hydrogel Scaffolds of Various Stiffnesses

NASA Astrophysics Data System (ADS)

Bhatnagar, Divya; Jurukovski, Vladimir; Rafailovich, Miriam; Simon, Marcia

2011-03-01

Dental Pulp Stem Cells (DPSCs) are known to differentiate in bone, dentine, or nerve tissue through different environment signals. This work investigates whether differentiation could occur in the absence of chemical induction and through mechanical stimuli only. For this study, we chose enzymatically cross-linked gelatin hydrogels as our substrates. Rheological studies carried out by oscillatory shear rheometry indicated that the modulus of the hardest hydrogel was of the order of 8kPa where as the medium and the softest hydrogel had modulus of the order of 1kPa and 100Pa respectively. DPSC were then plated on all three substrates and cultured with and without dexamethasone induction media. After 21 days of incubation, SEM analysis indicated that the cells cultured in the induction media produced biomineralized deposits on hard, medium as well as soft hydrogels. On the other hand, the cells cultured without the induction media also produced large amounts of biomineralized deposits.The modulus of the cells was also measured using AFM. En mass cell migration was also studied to determine the average velocity of migration of DPSCs. We also investigated whether stem cells that are induced to differentiate by their scaffold environment would continue to differentiate and biomineralize when removed from the inducing scaffold.

Hypothalamic neurogenesis persists in the aging brain and is controlled by energy-sensing IGF-I pathway.

PubMed

Chaker, Zayna; George, Caroline; Petrovska, Marija; Caron, Jean-Baptiste; Lacube, Philippe; Caillé, Isabelle; Holzenberger, Martin

2016-05-01

Hypothalamic tanycytes are specialized glial cells lining the third ventricle. They are recently identified as adult stem and/or progenitor cells, able to self-renew and give rise to new neurons postnatally. However, the long-term neurogenic potential of tanycytes and the pathways regulating lifelong cell replacement in the adult hypothalamus are largely unexplored. Using inducible nestin-CreER(T2) for conditional mutagenesis, we performed lineage tracing of adult hypothalamic stem and/or progenitor cells (HySC) and demonstrated that new neurons continue to be born throughout adult life. This neurogenesis was targeted to numerous hypothalamic nuclei and produced different types of neurons in the dorsal periventricular regions. Some adult-born neurons integrated the median eminence and arcuate nucleus during aging and produced growth hormone releasing hormone. We showed that adult hypothalamic neurogenesis was tightly controlled by insulin-like growth factors (IGF). Knockout of IGF-1 receptor from hypothalamic stem and/or progenitor cells increased neuronal production and enhanced α-tanycyte self-renewal, preserving this stem cell-like population from age-related attrition. Our data indicate that adult hypothalamus retains the capacity of cell renewal, and thus, a substantial degree of structural plasticity throughout lifespan. Copyright © 2016 Elsevier Inc. All rights reserved.

Proton beam writing of microstructures in Agar gel for patterned cell growth

NASA Astrophysics Data System (ADS)

Larisch, Wolfgang; Koal, Torsten; Werner, Ronald; Hohlweg, Marcus; Reinert, Tilo; Butz, Tilman

2011-10-01

A rather useful prerequisite for many biological and biophysical studies, e.g., for cell-cell communication or neuronal networks, is confined cell growth on micro-structured surfaces. Solidified Agar layers have smooth surfaces which are electrically neutral and thus inhibit receptor binding and cell adhesion. For the first time, Agar microstructures have been manufactured using proton beam writing (PBW). In the irradiated Agar material the polysaccharides are split into oligosaccharides which can easily be washed off leaving Agar-free areas for cell adhesion. The beam diameter of 1 μm allows the fabrication of compartments accommodating single cells which are connected by micrometer-sized channels. Using the external beam the production process is very fast. Up to 50 Petri dishes can be produced per day which makes this technique very suitable for biological investigations which require large throughputs.

Engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalyst for large scale biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid

PubMed Central

Seo, Joo-Hyun; Kim, Hwan-Hee; Jeon, Eun-Yeong; Song, Young-Ha; Shin, Chul-Soo; Park, Jin-Byung

2016-01-01

Baeyer-Villiger monooxygenases (BVMOs) are able to catalyze regiospecific Baeyer-Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are usually difficult to express in a functional form in microbial cells and are rather unstable under process conditions hindering their large-scale applications. Thereby, we investigated engineering of the BVMO from Pseudomonas putida KT2440 and the gene expression system to improve its activity and stability for large-scale biotransformation of ricinoleic acid (1) into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) (3), which can be hydrolyzed into 11-hydroxyundec-9-enoic acid (5) (i.e., a precursor of polyamide-11) and n-heptanoic acid (4). The polyionic tag-based fusion engineering of the BVMO and the use of a synthetic promoter for constitutive enzyme expression allowed the recombinant Escherichia coli expressing the BVMO and the secondary alcohol dehydrogenase of Micrococcus luteus to produce the ester (3) to 85 mM (26.6 g/L) within 5 h. The 5 L scale biotransformation process was then successfully scaled up to a 70 L bioreactor; 3 was produced to over 70 mM (21.9 g/L) in the culture medium 6 h after biotransformation. This study demonstrated that the BVMO-based whole-cell reactions can be applied for large-scale biotransformations. PMID:27311560

Physiological Effects of Free Fatty Acid Production in Genetically Engineered Synechococcus elongatus PCC 7942

PubMed Central

Ruffing, Anne M.; Jones, Howland D.T.

2012-01-01

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production. PMID:22473793

Antibody producing B lineage cells invade the central nervous system predominantly at the time of and triggered by acute Epstein-Barr virus infection: A hypothesis on the origin of intrathecal immunoglobulin synthesis in multiple sclerosis.

PubMed

Otto, Carolin; Hofmann, Jörg; Ruprecht, Klemens

2016-06-01

Patients with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), typically have an intrathecal synthesis of immunoglobulin (Ig)G. Intrathecal IgG is produced by B lineage cells that entered the CNS, but why and when these cells invade the CNS of patients with MS is unknown. The intrathecal IgG response in patients with MS is polyspecific and part of it is directed against different common viruses (e.g. measles virus, rubella virus, varicella zoster virus). Strong and consistent evidence suggests an association of MS and Epstein-Barr virus (EBV) infection and EBV seroprevalence in patients with MS is practically 100%. However, intriguingly, despite of the universal EBV seroprevalence, the frequency of intrathecally produced IgG to EBV in patients with MS is much lower than that of intrathecally produced IgG to other common viruses. The acute phase of primary EBV infection is characterized by a strong polyclonal B cell activation. As typical for humoral immune responses against viruses, EBV specific IgG is produced only with a temporal delay after acute EBV infection. Aiming to put the above facts into a logical structure, we here propose the hypothesis that in individuals going on to develop MS antibody producing B lineage cells invade the CNS predominantly at the time of and triggered by acute primary EBV infection. Because at the time of acute EBV infection EBV IgG producing B lineage cells have not yet occurred, the hypothesis could explain the universal EBV seroprevalence and the low frequency of intrathecally produced IgG to EBV in patients with MS. Evidence supporting the hypothesis could be provided by large prospective follow-up studies of individuals with symptomatic primary EBV infection (infectious mononucleosis). Furthermore, the clarification of the molecular mechanism underlying an EBV induced invasion of B lineage cells into the CNS of individuals going on to develop MS could corroborate it, too. If true, our hypothesis would link EBV infection, the most important environmental risk factor for MS, with intrathecal IgG synthesis, the most characteristic laboratory feature of MS. Besides explaining the origin of intrathecal IgG synthesis in patients with MS, the hypothesis could thus also provide a conceptual framework for clarifying the mechanism through which EBV contributes to the development of MS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phylogenetic Relationships of Yessotoxin-Producing Dinoflagellates, Based on the Large Subunit and Internal Transcribed Spacer Ribosomal DNA Domains▿

PubMed Central

Howard, Meredith D. A.; Smith, G. Jason; Kudela, Raphael M.

2009-01-01

Yessotoxin (YTX) is a globally distributed marine toxin produced by some isolates of the dinoflagellate species Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera within the order Gonyaulacales. The process of isolating cells and testing each isolate individually for YTX production during toxic blooms are labor intensive, and this impedes our ability to respond quickly to toxic blooms. In this study, we used molecular sequences from the large subunit and internal transcribed spacer genomic regions in the ribosomal operon of known YTX-producing dinoflagellates to determine if genetic differences exist among geographically distinct populations or between toxic and nontoxic isolates within species. In all analyses, all three YTX-producing species fell within the Gonyaulacales order in agreement with morphological taxonomy. Phylogenetic analyses of available rRNA gene sequences indicate that the capacity for YTX production appears to be confined to the order Gonyaulacales. These findings indicate that Gonyaulacoloid dinoflagellate species are the most likely to produce YTX and thus should be prioritized for YTX screening during events. Dinoflagellate species that fall outside of the Gonyaulacales order are unlikely to produce YTX. Although the rRNA operon offers multiple sequence domains to resolve species level diversification within this dinoflagellate order, these domains are not sufficiently variable to provide robust markers for YTX toxicity. PMID:19011074

Pin-Hole Free Perovskite Film for Solar Cells Application Prepared by Controlled Two-Step Spin-Coating Method

NASA Astrophysics Data System (ADS)

Bahtiar, A.; Rahmanita, S.; Inayatie, Y. D.

2017-05-01

Morphology of perovskite film is a key important for achieving high performance perovskite solar cells. Perovskite films are commonly prepared by two-step spin-coating method. However, pin-holes are frequently formed in perovskite films due to incomplete conversion of lead-iodide (PbI2) into perovskite CH3NH3PbI3. Pin-holes in perovskite film cause large hysteresis in current-voltage curve of solar cells due to large series resistance between perovskite layer-hole transport material. Moreover, crystal structure and grain size of perovskite crystal are also other important parameters for achieving high performance solar cells, which are significantly affected by preparation of perovskite film. We studied the effect of preparation of perovskite film using controlled spin-coating parameters on crystal structure and morphological properties of perovskite film. We used two-step spin-coating method for preparation of perovskite film with varied spinning speed, spinning time and temperature of spin-coating process to control growth of perovskite crystal aimed to produce high quality perovskite crystal with pin-hole free and large grain size. All experiment was performed in air with high humidity (larger than 80%). The best crystal structure, pin-hole free with large grain crystal size of perovskite film was obtained from film prepared at room temperature with spinning speed 1000 rpm for 20 seconds and annealed at 100°C for 300 seconds.

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation

PubMed Central

Cox, R F; Hernandez-Santana, A; Ramdass, S; McMahon, G; Harmey, J H; Morgan, M P

2012-01-01

Background: Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood. Methods: We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice. Results: Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice. Conclusion: This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development. PMID:22233923

The formation and dust lifting processes associated with a large Saharan meso-scale convective system (MCS)

NASA Astrophysics Data System (ADS)

Roberts, Alex; Knippertz, Peter

2013-04-01

This work focusses on the meteorology that produced a large Mesoscale Convective System (MCS) and the dynamics of its associated cold pool. The case occurred between 8th-10th June 2010 and was initiated over the Hoggar and Aïr Mountains in southern Algeria and northern Niger respectively. The dust plume created covered parts of Algeria, Mali and Mauritania and was later deformed the by background flow and transported over the Atlantic and Mediterranean. This study is based on: standard surface observations (where available), ERA-Interim reanalysis, Meteosat imagery, MODIS imagery, Tropical Rainfall Measuring Mission (TRMM) rainfall estimates, Cloud Aerosol Lidar and Infrared Pathfinder Satellite Observation (CALIPSO), CloudSat and a high resolution (3.3km) limited area simulation using the Weather Research and Forecasting (WRF) model. A variety of different processes appear to be important for the generation of this MCS and the spreading of the associated dusty cold pool. These include: the presence of a trough on the subtropical jet, the production of a tropical cloud plume, disruption to the structure of the Saharan heat low and the production of a Libyan high. These features produced moistening of the boundary layer and a convergence zone over the region of MCS initiation. Another important factor appears to have been the production of a smaller MCS and cold pool on the evening of the 7th June. This elevated low-level moisture and encouraged convective initiation the following day. Once triggered on the 8th June some cells grew and merged into a single large system that propagated south westward and produced a large cold pool that emanated from its northern edge. The cells on the northern edge of the system over the Hoggar grew and collapsed producing a haboob that spread over a large area. Cells further south continued to develop into the MCS and actively produce a cold pool over the system's lifetime. This undercut the dusty air from the earlier cold pool and forced dust high into the atmosphere. As well as the expected behaviour of a gravity current there also seems to be a complex relationship between the cold pool and diurnal variation in boundary layer structure. These include: (1) the production of nocturnal low-level jet in the area previously covered by the cold pool allowing for further dust uplift the following morning, (2) the development of a bore on the nocturnal boundary layer travelling ahead of the cold pool and capable of deflating dust further into the desert and (3) the production of bores on the nocturnal boundary layer by the collision of fronts formed through the collapse of the well mixed daytime boundary layer and nocturnal frontogenesis. It is hoped that this work will add to the understanding of the production of large Saharan MCSs and the processes that can influence their formation. Also it shows the complex dynamical interactions that occur within the Saharan boundary layer and how these might impact our understanding of dust uplift processes associated with the passage of MCSs.

Adult-specific insulin-producing neurons in Drosophila melanogaster.

PubMed

Ohhara, Yuya; Kobayashi, Satoru; Yamakawa-Kobayashi, Kimiko; Yamanaka, Naoki

2018-06-01

Holometabolous insects undergo metamorphosis to reorganize their behavioral and morphological features into adult-specific ones. In the central nervous system (CNS), some larval neurons undergo programmed cell death, whereas others go through remodeling of axonal and dendritic arbors to support functions of re-established adult organs. Although there are multiple neuropeptides that have stage-specific roles in holometabolous insects, the reorganization pattern of the entire neuropeptidergic system through metamorphosis still remains largely unclear. In this study, we conducted a mapping and lineage tracing of peptidergic neurons in the larval and adult CNS by using Drosophila genetic tools. We found that Diuretic hormone 44-producing median neurosecretory cells start expressing Insulin-like peptide 2 in the pharate adult stage. This neuronal cluster projects to the corpora cardiaca and dorsal vessel in both larval and adult stages, and also innervates an adult-specific structure in the digestive tract, the crop. We propose that the adult-specific insulin-producing cells may regulate functions of the digestive system in a stage-specific manner. Our study provides a neuroanatomical basis for understanding remodeling of the neuropeptidergic system during insect development and evolution. © 2018 Wiley Periodicals, Inc.

[Characteristics of growth and changes in the ultrafine structure of bacteria in the course of continuous cultivation on media containing ethanol].

PubMed

Kvasnikov, E I; Gavrilenko, M N; Sumnevich, V G; Stepaniuk, V V; Eluseeva, G S; Stognii, I P

1977-01-01

A large number of bacterial strains assimilating chemical ethanol has been isolated using an original technique. Active growth of strains belonging to the genera Pseudomonas and, particularly, Acinetobacter was registered on mineral media containing ethanol. A mathematical model was constructed select a strain of Acinetobacter calcoaceticus K-9 during its continuous cultivation on media containing ethanol. The model makes it possible to determine conditions for producing a present amount of the biomass, the percentage of its yield, and the produc;iveness as a function of the dilution rate, temperature, and the concentration of ethanol and phosphoric acid in the medium. The main characteristics of the growth process in the studied factor space were established. The optimum conditions were calculated for growth of the strain with respect to each of the criteria. Under various conditions of bacterial growth, changes in the morphology and ultra-fine structure of the cells correlated with their physiological activity. The volume of the cells increased with the rate of dilution of the medium: the process can be described by a saturation curve. The presence of mesosomal structures is typical of the cells growing at low flow rates.

Large Extremity Peripheral Nerve Repair

DTIC Science & Technology

2014-10-01

has also been shown to produce human-beta-3-defensin. These antimicrobial peptides are implicated in the resistance of epithelial surfaces to...gonadotrophin receptors that regulate prostaglandin production and activity. Epithelial cells manufacture multiple vasoactive peptides , growth factors...200734 P-RCT (n Z 102) PT burns Processed Amnion vs topical antimicrobials . Significantly less dressing changes with amnion. Time to healing, length

Cross reactive antibody and cytotoxic T lymphocytes from avian influenza H9N2 infected chickens against homologous and heterologous avian influenza isolates

USDA-ARS?s Scientific Manuscript database

Immunity against avian influenza (AI) is largely based on the induction of neutralizing antibodies produced against the hemagglutinin, although cytotoxic T lymphocytes (CTL’s) have been reported as critical for clearance of virus from infected cells. Antibody production against a particular virus ...

Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone.

PubMed

Aghili, Zahra Sadat; Zarkesh-Esfahani, Sayyed Hamid

2018-02-01

Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments.In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH.A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO 4 and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations. © Georg Thieme Verlag KG Stuttgart · New York.

Multi-casting approach for vascular networks in cellularized hydrogels.

PubMed

Justin, Alexander W; Brooks, Roger A; Markaki, Athina E

2016-12-01

Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. We report a new method for producing a hierarchical, three-dimensional (3D) and perfusable vasculature in a large, cellularized fibrin hydrogel. Bifurcating channels, varying in size from 1 mm to 200-250 µm, are formed using a novel process in which we convert a 3D printed thermoplastic material into a gelatin network template, by way of an intermediate alginate hydrogel. This enables a CAD-based model design, which is highly customizable, reproducible, and which can yield highly complex architectures, to be made into a removable material, which can be used in cellular environments. Our approach yields constructs with a uniform and high density of cells in the bulk, made from bioactive collagen and fibrin hydrogels. Using standard cell staining and immuno-histochemistry techniques, we showed good cell seeding and the presence of tight junctions between channel endothelial cells, and high cell viability and cell spreading in the bulk hydrogel. © 2016 The Authors.

Response Mechanisms in Serratia marcescens IBBPo15 During Organic Solvents Exposure.

PubMed

Stancu, Mihaela Marilena

2016-12-01

Serratia marcescens strain IBB Po15 (KT315653) which possesses serratiopeptidase (ser) gene (KT894207) exhibited good solvent tolerance. During the exposure of S. marcescens IBB Po15 cells to 5 % organic solvents, n-decane was less toxic for this bacterium, compared with n-hexane, cyclohexane, ethylbenzene, toluene, and styrene. The exposure of the S. marcescens IBB Po15 cells to n-hexane, cyclohexane, ethylbenzene, toluene, and styrene induced the formation of large clusters, while in control and n-decane-exposed cells, only organization into small clusters was observed. The data obtained suggested that S. marcescens IBB Po15 cells produced some secondary metabolites (i.e., surfactant serrawettin, red pigment prodigiosin) which are well known as valuable molecules due to their large applications. The exposure of the bacterial cells to organic solvents induced secondary metabolites profile modifications. However, S. marcescens IBB Po15 possesses only alkB1, todM, rhlAB, pswP, mpr, and ser genes, the unspecific amplification of other fragments being acquired also when the primers for alkM1, xylM, ndoM, and C23DO genes were used. Modifications of DNA patterns were not depicted in S. marcescens IBB Po15 cells exposed to organic solvents.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

PubMed

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Focused tandem shock waves in water and their potential application in cancer treatment

NASA Astrophysics Data System (ADS)

Lukes, P.; Sunka, P.; Hoffer, P.; Stelmashuk, V.; Pouckova, P.; Zadinova, M.; Zeman, J.; Dibdiak, L.; Kolarova, H.; Tomankova, K.; Binder, S.; Benes, J.

2014-01-01

The generator of two focused successive (tandem) shock waves (FTSW) in water produced by underwater multichannel electrical discharges at two composite electrodes, with a time delay between the first and second shock waves of 10 s, was developed. It produces, at the focus, a strong shock wave with a peak positive pressure of up to 80 MPa, followed by a tensile wave with a peak negative pressure of up to MPa, thus generating at the focus a large amount of cavitation. Biological effects of FTSW were demonstrated in vitro on hemolysis of erythrocytes and cell viability of human acute lymphoblastic leukemia cells as well as on tumor growth delay ex vivo and in vivo experiments performed with B16 melanoma, T-lymphoma, and R5-28 sarcoma cell lines. It was demonstrated in vivo that FTSW can enhance antitumor effects of chemotherapeutic drugs, such as cisplatin, most likely due to increased permeability of the membrane of cancer cells induced by FTSW. Synergetic cytotoxicity of FTSW with sonosensitive porphyrin-based drug Photosan on tumor growth was observed, possibly due to the cavitation-induced sonodynamic effect of FTSW.

Process for producing large grain cadmium telluride

DOEpatents

Hasoon, F.S.; Nelson, A.J.

1996-01-16

A process is described for producing a cadmium telluride polycrystalline film having grain sizes greater than about 20 {micro}m. The process comprises providing a substrate upon which cadmium telluride can be deposited and placing that substrate within a vacuum chamber containing a cadmium telluride effusion cell. A polycrystalline film is then deposited on the substrate through the steps of evacuating the vacuum chamber to a pressure of at least 10{sup {minus}6} torr.; heating the effusion cell to a temperature whereat the cell releases stoichiometric amounts of cadmium telluride usable as a molecular beam source for growth of grains on the substrate; heating the substrate to a temperature whereat a stoichiometric film of cadmium telluride can be deposited; and releasing cadmium telluride from the effusion cell for deposition as a film on the substrate. The substrate then is placed in a furnace having an inert gas atmosphere and heated for a sufficient period of time at an annealing temperature whereat cadmium telluride grains on the substrate grow to sizes greater than about 20 {micro}m.

Specification of functional cranial placode derivatives from human pluripotent stem cells.

PubMed

Dincer, Zehra; Piao, Jinghua; Niu, Lei; Ganat, Yosif; Kriks, Sonja; Zimmer, Bastian; Shi, Song-Hai; Tabar, Viviane; Studer, Lorenz

2013-12-12

Cranial placodes are embryonic structures essential for sensory and endocrine organ development. Human placode development has remained largely inaccessible despite the serious medical conditions caused by the dysfunction of placode-derived tissues. Here, we demonstrate the efficient derivation of cranial placodes from human pluripotent stem cells. Timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Concomitant inhibition of fibroblast growth factor signaling disrupts placode derivation and induces surface ectoderm. Further fate specification at the preplacode stage enables the selective generation of placode-derived trigeminal ganglia capable of in vivo engraftment, mature lens fibers, and anterior pituitary hormone-producing cells that upon transplantation produce human growth hormone and adrenocorticotropic hormone in vivo. Our results establish a powerful experimental platform to study human cranial placode development and set the stage for the development of human cell-based therapies in sensory and endocrine disease. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

One step screening of retroviral producer clones by real time quantitative PCR.

PubMed

Towers, G J; Stockholm, D; Labrousse-Najburg, V; Carlier, F; Danos, O; Pagès, J C

1999-01-01

Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.

Adoptive Transfer of Tumor-Specific Th2 Cells Eradicates Tumors by Triggering an In Situ Inflammatory Immune Response.

PubMed

Lorvik, Kristina Berg; Hammarström, Clara; Fauskanger, Marte; Haabeth, Ole Audun Werner; Zangani, Michael; Haraldsen, Guttorm; Bogen, Bjarne; Corthay, Alexandre

2016-12-01

Adoptive cell therapy (ACT) trials to date have focused on transfer of autologous tumor-specific cytotoxic CD8 + T cells; however, the potential of CD4 + T helper (Th) cells for ACT is gaining interest. While encouraging results have been reported with IFNγ-producing Th1 cells, tumor-specific Th2 cells have been largely neglected for ACT due to their reported tumor-promoting properties. In this study, we tested the efficacy of idiotype-specific Th2 cells for the treatment of mice with MHC class II-negative myeloma. Th2 ACT efficiently eradicated subcutaneous myeloma in an antigen-specific fashion. Transferred Th2 cells persisted in vivo and conferred long-lasting immunity. Cancer eradication mediated by tumor-specific Th2 cells did not require B cells, natural killer T cells, CD8 + T cells, or IFNγ. Th2 ACT was also curative against B-cell lymphoma. Upon transfer, Th2 cells induced a type II inflammation at the tumor site with massive infiltration of M2-type macrophages producing arginase. In vivo blockade of arginase strongly inhibited Th2 ACT, consistent with a key role of arginase and M2 macrophages in myeloma elimination by Th2 cells. These results illustrate that cancer eradication may be achieved by induction of a tumor-specific Th2 inflammatory immune response at the tumor site. Thus, ACT with tumor-specific Th2 cells may represent a highly efficient immunotherapy protocol against cancer. Cancer Res; 76(23); 6864-76. ©2016 AACR. ©2016 American Association for Cancer Research.

Cell fusion and nuclear fusion in plants.

PubMed

Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

2016-12-01

Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

Why plants make puzzle cells, and how their shape emerges.

PubMed

Sapala, Aleksandra; Runions, Adam; Routier-Kierzkowska, Anne-Lise; Das Gupta, Mainak; Hong, Lilan; Hofhuis, Hugo; Verger, Stéphane; Mosca, Gabriella; Li, Chun-Biu; Hay, Angela; Hamant, Olivier; Roeder, Adrienne Hk; Tsiantis, Miltos; Prusinkiewicz, Przemyslaw; Smith, Richard S

2018-02-27

The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis. © 2018, Sapala et al.

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system.

PubMed

Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

2018-04-01

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system

NASA Astrophysics Data System (ADS)

Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

2018-04-01

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform.

Why plants make puzzle cells, and how their shape emerges

PubMed Central

Routier-Kierzkowska, Anne-Lise; Das Gupta, Mainak; Hong, Lilan; Hofhuis, Hugo; Verger, Stéphane; Mosca, Gabriella; Li, Chun-Biu; Hay, Angela; Hamant, Olivier; Roeder, Adrienne HK; Tsiantis, Miltos; Prusinkiewicz, Przemyslaw

2018-01-01

The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis. PMID:29482719

CD4+ T helper 2 cells – microbial triggers, differentiation requirements and effector functions

PubMed Central

Okoye, Isobel S; Wilson, Mark S

2011-01-01

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate-like cells with the capacity to secrete large amounts of interleukin-5 (IL-5), IL-13 and IL-9 as well as IL-4-producing and antigen-processing basophils have (re)-emerged onto the type 2 scene. To what extent these new players influence αβ+ CD4+ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T-cell receptor ligation, co-stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned. PMID:22043920

Counting cell number in situ by quantification of dimethyl sulphide in culture headspace.

PubMed

Chippendale, Thomas W E; Španěl, Patrik; Smith, David; El Haj, Alicia J

2014-10-07

A novel, non-invasive technique is reported for determining the numbers of cells in a culture by quantifying dimethyl sulphide (DMS) in the culture headspace as produced by the cellular enzymatic reduction of dissolved dimethyl sulphoxide (DMSO). Measured DMS concentrations, as performed using selected ion flow tube mass spectrometry (SIFT-MS), in the headspace of 2D and 3D cultures of four cell lines, viz. HEK293 (kidney), MG63 (bone), hepG2 (liver) and CALU-1 (lung), linearly correlate with starting cell number. Clear differences in the rates of production of DMS by the four cell types in both the 2D and 3D situations are seen. This novel analytical technique for cell enumeration offers a significant contribution to quality assessment across cell-based research and industry, including analysis of large scale culture systems, and for routine cell biology research.

A Novel Wounding Device Suitable for Quantitative Biochemical Analysis of Wound Healing and Regeneration of Cultured Epithelium

PubMed Central

Lan, Rongpei; Geng, Hui; Hwang, Yoon; Mishra, Pramod; Skloss, Wayne L.; Sprague, Eugene A.; Saikumar, Pothana; Venkatachalam, Manjeri

2010-01-01

We describe the fabrication and use of an in vitro wounding device that denudes cultured epithelium in patterns designed to leave behind strips or islands of cells sufficiently narrow or small to ensure that all remaining cells become rapidly activated and then migrate, dedifferentiate and proliferate in near synchrony. The design ensures that signals specific to regenerating cells do not become diluted by quiescent differentiated cells that are not affected by wound induced activation. The device consists of a flat circular disk of rubber engraved to produce alternating ridges and grooves in patterns of concentric circles or parallel lines. The disk is mounted at the end of a pneumatically controlled piston assembly. Application of controlled pressure and circular or linear movement of the disk on cultures produced highly reproducible wounding patterns. The near synchronous regenerative activity of cell bands or islands permitted the collection of samples large enough for biochemical studies to sensitively detect alterations involving mRNA for several early response genes and protein phosphorylation in major signaling pathways. The method is versatile, easy to use and reproducible, and should facilitate biochemical, proteomic and genomic studies of wound induced regeneration of cultured epithelium. PMID:20230600

Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective

PubMed Central

Bryan, Daniel; El-Shibiny, Ayman; Hobbs, Zack; Porter, Jillian; Kutter, Elizabeth M.

2016-01-01

Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. “Hibernation” mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a “scavenger” response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems able to produce no more than an average of one phage per originally available cell, and few if any further progeny are produced by cells in this mode even if fresh nutrients are made available later. PMID:27660625

Hot cell purification of strontium-82, 85 and other isotopes from proton irradiated molybdenum

DOEpatents

Bentley, G.E.; Barnes, J.W.

1979-10-17

A process suitable for producing curie quantities of quite pure Sr-82,85 is given. After a Mo target is irradiated with energetic protons having energies greater than about 200 MeV, thus producing a large number of radioactive species, the particular species of Sr-82,85 are substantially separated from the other products by a 6-step process. The process comprises dissolution of the target in H/sub 2/O/sub 2/, followed by use of several ion exchange resins, extraction with an organophosphorus compound, and several adjustments of pH values. Other embodiments include processes for producing relatively pure long-lived Rb isotopes, Y-88, and Zr-88.

Hot cell purification of strontium-82, 85 and other isotopes from proton irradiated molybdenum

DOEpatents

Bentley, Glenn E.; Barnes, John W.

1981-01-01

A process suitable for producing curie quantities of quite pure Sr-82,85 is given. After a Mo target is irradiated with energetic protons having energies greater than about 200 MeV, thus producing a large number of radioactive species, the particular species of Sr-82,85 are substantially separated from the other products by a 6-step process. The process comprises dissolution of the target in H.sub.2 O.sub.2, followed by use of several ion exchange resins, extraction with an organophosphorus compound, and several adjustments of pH values. Other embodiments include processes for producing relatively pure long-lived Rb isotopes, Y-88, and Zr-88.

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

PubMed Central

Ohyama, Chikara; Kanto, Satoru; Kato, Kazunori; Nakano, Osamu; Arai, Yoichi; Kato, Tetsuro; Chen, Shihao; Fukuda, Michiko N.; Fukuda, Minoru

2002-01-01

Epithelial carcinoma and leukemia cells express sialyl Lewis x oligosaccharides as tumor-associated carbohydrate antigens. To determine the role of sialyl Lewis x oligosaccharides in tumor dissemination, human melanoma MeWo cells, which do not express sialyl Lewis x, were transfected with α1,3-fucosyltransferase III (FTIII), and cell lines expressing different amounts of sialyl Lewis x were isolated. When these cells were injected into the tail vein of nude mice, cells expressing moderate amounts of sialyl Lewis x (MeWo-FTIII⋅M) produced a significantly greater number of lung tumor foci than did parental MeWo cells. In contrast, cells expressing large amounts of sialyl Lewis x (MeWo-FTIII⋅H) produced few lung tumor foci in nude mice but were highly tumorigenic in beige mice, which have defective natural killer (NK) cells. In vitro assays demonstrated that MeWo-FTIII⋅H cells are much more sensitive to NK cell-mediated cytotoxicity than are MeWo-FTIII⋅M cells or parental MeWo cells and the susceptibility of MeWo-FTIII⋅H cells to NK cell-mediated cytolysis can be inhibited by preincubating MeWo-FTIII⋅H cells with anti-sialyl Lewis x antibody. Moreover, we discovered that NK cell-mediated cytolysis of MeWo-FTIII⋅H cells can be inhibited by the addition of an antibody against the NK cell receptor CD94 or sialyl Lewis x oligosaccharides. These results, combined with structural analysis of MeWo-FTIII⋅H cell carbohydrates, indicate that moderate amounts of sialyl Lewis x lead to tumor metastasis, whereas expression of high levels of sialyl Lewis x leads to an NK cell attack on tumor cells, demonstrating that expression of different amounts of sialyl Lewis x results in entirely different biological consequences. PMID:12370411

A Life-Cycle Model of Human Social Groups Produces a U-Shaped Distribution in Group Size.

PubMed

Salali, Gul Deniz; Whitehouse, Harvey; Hochberg, Michael E

2015-01-01

One of the central puzzles in the study of sociocultural evolution is how and why transitions from small-scale human groups to large-scale, hierarchically more complex ones occurred. Here we develop a spatially explicit agent-based model as a first step towards understanding the ecological dynamics of small and large-scale human groups. By analogy with the interactions between single-celled and multicellular organisms, we build a theory of group lifecycles as an emergent property of single cell demographic and expansion behaviours. We find that once the transition from small-scale to large-scale groups occurs, a few large-scale groups continue expanding while small-scale groups gradually become scarcer, and large-scale groups become larger in size and fewer in number over time. Demographic and expansion behaviours of groups are largely influenced by the distribution and availability of resources. Our results conform to a pattern of human political change in which religions and nation states come to be represented by a few large units and many smaller ones. Future enhancements of the model should include decision-making rules and probabilities of fragmentation for large-scale societies. We suggest that the synthesis of population ecology and social evolution will generate increasingly plausible models of human group dynamics.

A Life-Cycle Model of Human Social Groups Produces a U-Shaped Distribution in Group Size

PubMed Central

Salali, Gul Deniz; Whitehouse, Harvey; Hochberg, Michael E.

2015-01-01

One of the central puzzles in the study of sociocultural evolution is how and why transitions from small-scale human groups to large-scale, hierarchically more complex ones occurred. Here we develop a spatially explicit agent-based model as a first step towards understanding the ecological dynamics of small and large-scale human groups. By analogy with the interactions between single-celled and multicellular organisms, we build a theory of group lifecycles as an emergent property of single cell demographic and expansion behaviours. We find that once the transition from small-scale to large-scale groups occurs, a few large-scale groups continue expanding while small-scale groups gradually become scarcer, and large-scale groups become larger in size and fewer in number over time. Demographic and expansion behaviours of groups are largely influenced by the distribution and availability of resources. Our results conform to a pattern of human political change in which religions and nation states come to be represented by a few large units and many smaller ones. Future enhancements of the model should include decision-making rules and probabilities of fragmentation for large-scale societies. We suggest that the synthesis of population ecology and social evolution will generate increasingly plausible models of human group dynamics. PMID:26381745

Preparation and characterization of monoclonal antibody against digoxin.

PubMed

Kashanian, S; Rasaee, M J; Paknejad, M; Omidfar, K; Pour-Amir, M; Rajabi, Bazl M

2002-10-01

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

PubMed Central

Kallio, A; Jänne, J

1983-01-01

Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone. PMID:6411077

Changes in E-cadherin rigidity sensing regulate cell adhesion.

PubMed

Collins, Caitlin; Denisin, Aleksandra K; Pruitt, Beth L; Nelson, W James

2017-07-18

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform | Office of Cancer Genomics

Cancer.gov

The vast datasets generated by next generation gene sequencing and expression profiling have transformed biological and translational research. However, technologies to produce large-scale functional genomics datasets, such as high-throughput detection of protein-protein interactions (PPIs), are still in early development. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform featured with both high sensitivity and robustness in a mammalian cell environment remains to be established.

An avian cell line designed for production of highly attenuated viruses.

PubMed

Jordan, Ingo; Vos, Ad; Beilfuss, Stefanie; Neubert, Andreas; Breul, Sabine; Sandig, Volker

2009-01-29

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

Fragmentation scaling of percolation clusters in two and three dimensions: Large-cell Monte Carlo RG approach

NASA Astrophysics Data System (ADS)

Cheon, M.; Chang, I.

1999-04-01

The scaling behavior for a binary fragmentation of critical percolation clusters is investigated by a large-cell Monte Carlo real-space renormalization group method in two and three dimensions. We obtain accurate values of critical exponents λ and phi describing the scaling of fragmentation rate and the distribution of fragments' masses produced by a binary fragmentation. Our results for λ and phi show that the fragmentation rate is proportional to the size of mother cluster, and the scaling relation σ = 1 + λ - phi conjectured by Edwards et al. to be valid for all dimensions is satisfied in two and three dimensions, where σ is the crossover exponent of the average cluster number in percolation theory, which excludes the other scaling relations.

Colonization of bone matrices by cellular components

NASA Astrophysics Data System (ADS)

Shchelkunova, E. I.; Voropaeva, A. A.; Korel, A. V.; Mayer, D. A.; Podorognaya, V. T.; Kirilova, I. A.

2017-09-01

Practical surgery, traumatology, orthopedics, and oncology require bioengineered constructs suitable for replacement of large-area bone defects. Only rigid/elastic matrix containing recipient's bone cells capable of mitosis, differentiation, and synthesizing extracellular matrix that supports cell viability can comply with these requirements. Therefore, the development of the techniques to produce structural and functional substitutes, whose three-dimensional structure corresponds to the recipient's damaged tissues, is the main objective of tissue engineering. This is achieved by developing tissue-engineering constructs represented by cells placed on the matrices. Low effectiveness of carrier matrix colonization with cells and their uneven distribution is one of the major problems in cell culture on various matrixes. In vitro studies of the interactions between cells and material, as well as the development of new techniques for scaffold colonization by cellular components are required to solve this problem.

Clinical Application of Stem Cells in the Cardiovascular System

NASA Astrophysics Data System (ADS)

Stamm, Christof; Klose, Kristin; Choi, Yeong-Hoon

Regenerative medicine encompasses "tissue engineering" - the in vitro fabrication of tissues and/or organs using scaffold material and viable cells - and "cell therapy" - the transplantation or manipulation of cells in diseased tissue in vivo. In the cardiovascular system, tissue engineering strategies are being pursued for the development of viable replacement blood vessels, heart valves, patch material, cardiac pacemakers and contractile myocardium. Anecdotal clinical applications of such vessels, valves and patches have been described, but information on systematic studies of the performance of such implants is not available, yet. Cell therapy for cardiovascular regeneration, however, has been performed in large series of patients, and numerous clinical studies have produced sometimes conflicting results. The purpose of this chapter is to summarize the clinical experience with cell therapy for diseases of the cardiovascular system, and to analyse possible factors that may influence its outcome.

Comparison of SensL and Hamamatsu 4×4 channel SiPM arrays in gamma spectrometry with scintillators

NASA Astrophysics Data System (ADS)

Grodzicka-Kobylka, M.; Szczesniak, T.; Moszyński, M.

2017-06-01

The market of Silicon Photomultipliers (SiPMs) consists of many manufacturers that produce their detectors in different technology. Hamamatsu (Japan) and SensL (Ireland) seems to be the most popular companies that produce large SiPM arrays. The aim of this work is characterization and comparison of 4×4 channel SiPM arrays produced by these two producers. Both of the tested SiPMs are made in through-silicon via (TSV) technology, consist of 16, 3×3 mm avalanche photodiode (APD) cells and have fill factor slightly above 60%. The largest difference is a single APD cell size and hence total number of APD cells (55,424 for Hamamatsu, 76,640 for SensL). In the case of SensL SiPM, its spectral response characteristics is shifted slightly toward shorter wavelengths with maximum at 420 nm (450 nm for Hamamatsu). The presented measurements cover selection of the SiPM optimum operating voltage (in respect to energy resolution), verification of the excess noise factor and check of the linearity characteristics. Moreover, the gamma spectrometry with LSO, BGO and CsI:Tl scintillators together with pulse characteristics for these crystals (rise time and fall time) is reported, as well as temperature dependence. The presented measurements show better performance of the SensL array comparing to the Hamamatsu detector.

Enrichment of IFN-γ producing cells in different murine adipose tissue depots upon infection with an apicomplexan parasite

PubMed Central

Teixeira, Luzia; Marques, Raquel M.; Ferreirinha, Pedro; Bezerra, Filipa; Melo, Joana; Moreira, João; Pinto, Ana; Correia, Alexandra; Ferreira, Paula G.; Vilanova, Manuel

2016-01-01

Here we report that lean mice infected with the intracellular parasite Neospora caninum show a fast but sustained increase in the frequency of IFN-γ-producing cells noticeable in distinct adipose tissue depots. Moreover, IFN-γ-mediated immune memory could be evoked in vitro in parasite antigen-stimulated adipose tissue stromal vascular fraction cells collected from mice infected one year before. Innate or innate-like cells such as NK, NK T and TCRγδ+ cells, but also CD4+ and CD8+ TCRβ+ lymphocytes contributed to the IFN-γ production observed since day one of infection. This early cytokine production was largely abrogated in IL-12/IL23 p40-deficient mice. Moreover, production of IFN-γ by stromal vascular fraction cells isolated from these mice was markedly lower than that of wild-type counterparts upon stimulation with parasite antigen. In wild-type mice the increased IFN-γ production was concomitant with up-regulated expression of genes encoding interferon-inducible GTPases and nitric oxide synthase, which are important effector molecules in controlling intracellular parasite growth. This increased gene expression was markedly impaired in the p40-deficient mice. Overall, these results show that NK cells but also diverse T cell populations mediate a prompt and widespread production of IFN-γ in the adipose tissue of N. caninum infected mice. PMID:27001522

Small phytoplankton and carbon export from the surface ocean.

PubMed

Richardson, Tammi L; Jackson, George A

2007-02-09

Autotrophic picoplankton dominate primary production over large oceanic regions but are believed to contribute relatively little to carbon export from surface layers. Using analyses of data from the equatorial Pacific Ocean and Arabian Sea, we show that the relative direct and indirect contribution of picoplankton to export is proportional to their total net primary production, despite their small size. We suggest that all primary producers, not just the large cells, can contribute to export from the surface layer of the ocean at rates proportional to their production rates.

The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells.

PubMed

Park, Ji-Yun; Shin, Dong-Jun; Lee, Soo-Hyeon; Lee, Je-Jung; Suh, Guk-Hyun; Cho, Duck; Kim, Sang-Ki

2015-04-17

Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3-CD5-CD21- NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Corning HYPERFlask® for viral amplification and production of diagnostic reagents.

PubMed

Kearney, Brian J; Voorhees, Matthew A; Williams, Priscilla L; Olschner, Scott P; Rossi, Cynthia A; Schoepp, Randal J

2017-04-01

Viral preparations are essential components in diagnostic research and development. The production of large quantities of virus traditionally is done by infecting numerous tissue culture flasks or roller bottles, which require large incubators and/or roller bottle racks. The Corning HYPERFlask ® is a multilayer flask that uses a gas permeable film to provide gas exchange between the cells and culture medium and the atmospheric environment. This study evaluated the suitability of the HYPERFlask for production of Lassa, Ebola, Bundibugyo, Reston, and Marburg viruses and compared it to more traditional methods using tissue culture flasks and roller bottles. The HYPERFlask produced cultures were equivalent in virus titer and indistinguishable in immunodiagnostic assays. The use of the Corning HYPERFlask for viral production is a viable alternative to traditional tissue culture flasks and roller bottles. HYPERFlasks allow for large volumes of virus to be produced in a small space without specialized equipment. Copyright © 2016. Published by Elsevier B.V.

TNF-induced necroptosis requires the plasma membrane localization of the MLKL protein | Center for Cancer Research

Cancer.gov

The cell signaling protein tumor necrosis factor (TNF), produced by white blood cells, promotes inflammation and immunity processes such as fever and is involved in tumorigenesis and apoptosis (programmed cell death). However, dysregulation of TNF can also lead to another form of programmed cell death called necroptosis, which is characterized by a rise in intracellular Ca2+, generation of reactive oxygen species (ROS), intracellular acidity, depletion of ATP, and, eventually, plasma membrane rupture. TNF-induced necroptosis has been associated with a wide variety of diseases including neurodegenerative diseases, major depression, rheumatoid arthritis, and cancer. Whereas the signaling mechanisms underlying TNF-induced apoptosis have largely been determined, the events precipitating in TNF-initiated necroptosis are still unknown.

Structures and properties of naturally occurring polyether antibiotics.

PubMed

Rutkowski, Jacek; Brzezinski, Bogumil

2013-01-01

Polyether ionophores represent a large group of natural, biologically active substances produced by Streptomyces spp. They are lipid soluble and able to transport metal cations across cell membranes. Several of polyether ionophores are widely used as growth promoters in veterinary. Polyether antibiotics show a broad spectrum of bioactivity ranging from antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Recently, it has been shown that some of these compounds are able to selectively kill cancer stem cells and multidrug-resistant cancer cells. Thus, they are recognized as new potential anticancer drugs. The biological activity of polyether ionophores is strictly connected with their molecular structure; therefore, the purpose of this paper is to present an overview of their formula, molecular structure, and properties.

Structures and Properties of Naturally Occurring Polyether Antibiotics

PubMed Central

Rutkowski, Jacek; Brzezinski, Bogumil

2013-01-01

Polyether ionophores represent a large group of natural, biologically active substances produced by Streptomyces spp. They are lipid soluble and able to transport metal cations across cell membranes. Several of polyether ionophores are widely used as growth promoters in veterinary. Polyether antibiotics show a broad spectrum of bioactivity ranging from antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Recently, it has been shown that some of these compounds are able to selectively kill cancer stem cells and multidrug-resistant cancer cells. Thus, they are recognized as new potential anticancer drugs. The biological activity of polyether ionophores is strictly connected with their molecular structure; therefore, the purpose of this paper is to present an overview of their formula, molecular structure, and properties. PMID:23586016

Chronic Fibro-Inflammatory Responses in Autoimmune Pancreatitis Depend on IFN-α and IL-33 Produced by Plasmacytoid Dendritic Cells.

PubMed

Watanabe, Tomohiro; Yamashita, Kouhei; Arai, Yasuyuki; Minaga, Kosuke; Kamata, Ken; Nagai, Tomoyuki; Komeda, Yoriaki; Takenaka, Mamoru; Hagiwara, Satoru; Ida, Hiroshi; Sakurai, Toshiharu; Nishida, Naoshi; Strober, Warren; Kudo, Masatoshi

2017-05-15

In previous studies, we found that human IgG4-related autoimmune pancreatitis (AIP) and murine AIP are driven by activation of plasmacytoid dendritic cells (pDCs) producing IFN-α. In the present studies we examined additional roles of pDC-related mechanisms in AIP pathogenesis, particularly those responsible for induction of fibrosis. We found that in murine AIP (MRL/Mp mice treated with polyinosinic-polycytidylic acid) not only the pancreatic infiltration of immune cells but also the development of fibrosis were markedly reduced by the depletion of pDCs or blockade of type I IFN signaling; moreover, such treatment was accompanied by a marked reduction of pancreatic expression of IL-33. Conversely, polyinosinic-polycytidylic acid-induced inflamed pancreatic tissue in murine AIP exhibited increased expression of type I IFNs and IL-33 (and downstream IL-33 cytokines such as IL-13 and TGF-β1). pDCs stimulated by type I IFN were the source of the IL-33 because purified populations of these cells isolated from the inflamed pancreas produced a large amount of IL-33 upon activation by TLR9 ligands, and such production was abrogated by the neutralization of type I IFN. The role of IL-33 in murine AIP pathogenesis was surprisingly important because blockade of IL-33 signaling by anti-ST2 Ab attenuated both pancreatic inflammation and accompanying fibrosis. Finally, whereas patients with both conventional pancreatitis and IgG4-related AIP exhibited increased numbers of acinar cells expressing IL-33, only the latter also exhibited pDCs producing this cytokine. These data thus suggest that pDCs producing IFN-α and IL-33 play a pivotal role in the chronic fibro-inflammatory responses underlying murine AIP and human IgG4-related AIP. Copyright © 2017 by The American Association of Immunologists, Inc.

Contribution of extracellular ATP on the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation.

PubMed

Kita, Toshiyuki; Arakaki, Naokatu

2015-01-01

Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and β subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.

3D-Printed ABS and PLA Scaffolds for Cartilage and Nucleus Pulposus Tissue Regeneration.

PubMed

Rosenzweig, Derek H; Carelli, Eric; Steffen, Thomas; Jarzem, Peter; Haglund, Lisbet

2015-07-03

Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.

Laser surface treatment of porous ceramic substrate for application in solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Mahmod, D. S. A.; Khan, A. A.; Munot, M. A.; Glandut, N.; Labbe, J. C.

2016-08-01

Laser has offered a large number of benefits for surface treatment of ceramics due to possibility of localized heating, very high heating/cooling rates and possibility of growth of structural configurations only produced under non-equilibrium high temperature conditions. The present work investigates oxidation of porous ZrB2-SiC sintered ceramic substrates through treatment by a 1072 ± 10 nm ytterbium fiber laser. A multi-layer structure is hence produced showing successively oxygen rich distinct layers. The porous bulk beneath these layers remained unaffected as this laser-formed oxide scale and protected the substrate from oxidation. A glassy SiO2 structure thus obtained on the surface of the substrate becomes subject of interest for further research, specifically for its utilization as solid protonic conductor in Solid Oxide Fuel Cells (SOFCs).

Evolution of plant conducting cells: perspectives from key regulators of vascular cell differentiation.

PubMed

Ohtani, Misato; Akiyoshi, Nobuhiro; Takenaka, Yuto; Sano, Ryosuke; Demura, Taku

2017-01-01

One crucial problem that plants faced during their evolution, particularly during the transition to growth on land, was how to transport water, nutrients, metabolites, and small signaling molecules within a large, multicellular body. As a solution to this problem, land plants developed specific tissues for conducting molecules, called water-conducting cells (WCCs) and food-conducting cells (FCCs). The well-developed WCCs and FCCs in extant plants are the tracheary elements and sieve elements, respectively, which are found in vascular plants. Recent molecular genetic studies revealed that transcriptional networks regulate the differentiation of tracheary and sieve elements, and that the networks governing WCC differentiation are largely conserved among land plant species. In this review, we discuss the molecular evolution of plant conducting cells. By focusing on the evolution of the key transcription factors that regulate vascular cell differentiation, the NAC transcription factor VASCULAR-RELATED NAC-DOMAIN for WCCs and the MYB-coiled-coil (CC)-type transcription factor ALTERED PHLOEM DEVELOPMENT for sieve elements, we describe how land plants evolved molecular systems to produce the specialized cells that function as WCCs and FCCs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

PubMed Central

Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

2015-01-01

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

PubMed

Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

2015-10-09

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

Prediction of anti-cancer drug response by kernelized multi-task learning.

PubMed

Tan, Mehmet

2016-10-01

Chemotherapy or targeted therapy are two of the main treatment options for many types of cancer. Due to the heterogeneous nature of cancer, the success of the therapeutic agents differs among patients. In this sense, determination of chemotherapeutic response of the malign cells is essential for establishing a personalized treatment protocol and designing new drugs. With the recent technological advances in producing large amounts of pharmacogenomic data, in silico methods have become important tools to achieve this aim. Data produced by using cancer cell lines provide a test bed for machine learning algorithms that try to predict the response of cancer cells to different agents. The potential use of these algorithms in drug discovery/repositioning and personalized treatments motivated us in this study to work on predicting drug response by exploiting the recent pharmacogenomic databases. We aim to improve the prediction of drug response of cancer cell lines. We propose to use a method that employs multi-task learning to improve learning by transfer, and kernels to extract non-linear relationships to predict drug response. The method outperforms three state-of-the-art algorithms on three anti-cancer drug screen datasets. We achieved a mean squared error of 3.305 and 0.501 on two different large scale screen data sets. On a recent challenge dataset, we obtained an error of 0.556. We report the methodological comparison results as well as the performance of the proposed algorithm on each single drug. The results show that the proposed method is a strong candidate to predict drug response of cancer cell lines in silico for pre-clinical studies. The source code of the algorithm and data used can be obtained from http://mtan.etu.edu.tr/Supplementary/kMTrace/. Copyright © 2016 Elsevier B.V. All rights reserved.

The effect of cell phones on human health

NASA Astrophysics Data System (ADS)

Abu-Isbeih, Ibrahim N.; Saad, Dina

2011-10-01

The effect of cell phone radiation on human health is the subject of recent interest and study, as a result of the enormous increase in cell phone usage throughout the world. Cell phones use electromagnetic radiation in the microwave range, which some believe may be harmful to human health. Other digital wireless systems, such as data communication networks, produce similar radiation. The objective of this survey is to review the effects of cell phones on human health: A large body of research exists, both epidemiological and experimental, in non-human animals and in humans, of which the majority shows no definite causative relationship between exposure to cell phones and harmful biological effects in humans. This is often paraphrased simply as the balance of evidence showing no harm to humans from cell phones, although a significant number of individual studies do suggest such a relationship, or are inconclusive.

Photoelectrochemical water splitting in separate oxygen and hydrogen cells

NASA Astrophysics Data System (ADS)

Landman, Avigail; Dotan, Hen; Shter, Gennady E.; Wullenkord, Michael; Houaijia, Anis; Maljusch, Artjom; Grader, Gideon S.; Rothschild, Avner

2017-06-01

Solar water splitting provides a promising path for sustainable hydrogen production and solar energy storage. One of the greatest challenges towards large-scale utilization of this technology is reducing the hydrogen production cost. The conventional electrolyser architecture, where hydrogen and oxygen are co-produced in the same cell, gives rise to critical challenges in photoelectrochemical water splitting cells that directly convert solar energy and water to hydrogen. Here we overcome these challenges by separating the hydrogen and oxygen cells. The ion exchange in our cells is mediated by auxiliary electrodes, and the cells are connected to each other only by metal wires, enabling centralized hydrogen production. We demonstrate hydrogen generation in separate cells with solar-to-hydrogen conversion efficiency of 7.5%, which can readily surpass 10% using standard commercial components. A basic cost comparison shows that our approach is competitive with conventional photoelectrochemical systems, enabling safe and potentially affordable solar hydrogen production.

Membrane potential dynamics of grid cells

PubMed Central

Domnisoru, Cristina; Kinkhabwala, Amina A.; Tank, David W.

2014-01-01

During navigation, grid cells increase their spike rates in firing fields arranged on a strikingly regular triangular lattice, while their spike timing is often modulated by theta oscillations. Oscillatory interference models of grid cells predict theta amplitude modulations of membrane potential during firing field traversals, while competing attractor network models predict slow depolarizing ramps. Here, using in-vivo whole-cell recordings, we tested these models by directly measuring grid cell intracellular potentials in mice running along linear tracks in virtual reality. Grid cells had large and reproducible ramps of membrane potential depolarization that were the characteristic signature tightly correlated with firing fields. Grid cells also exhibited intracellular theta oscillations that influenced their spike timing. However, the properties of theta amplitude modulations were not consistent with the view that they determine firing field locations. Our results support cellular and network mechanisms in which grid fields are produced by slow ramps, as in attractor models, while theta oscillations control spike timing. PMID:23395984

Concise Review: Parthenote Stem Cells for Regenerative Medicine: Genetic, Epigenetic, and Developmental Features

PubMed Central

Daughtry, Brittany

2014-01-01

Embryonic stem cells (ESCs) have the potential to provide unlimited cells and tissues for regenerative medicine. ESCs derived from fertilized embryos, however, will most likely be rejected by a patient’s immune system unless appropriately immunomatched. Pluripotent stem cells (PSCs) genetically identical to a patient can now be established by reprogramming of somatic cells. However, practical applications of PSCs for personalized therapies are projected to be unfeasible because of the enormous cost and time required to produce clinical-grade cells for each patient. ESCs derived from parthenogenetic embryos (pESCs) that are homozygous for human leukocyte antigens may serve as an attractive alternative for immunomatched therapies for a large population of patients. In this study, we describe the biology and genetic nature of mammalian parthenogenesis and review potential advantages and limitations of pESCs for cell-based therapies. PMID:24443005

Salt-mediated multicell formation in Deinococcus radiodurans.

PubMed Central

Chou, F I; Tan, S T

1991-01-01

The highly radiation-resistant tetracoccal bacterium Deinococcus radiodurans exhibited a reversible multi-cell-form transition which depended on the NaCl concentration in the medium. In response to 0.8% NaCl addition into the medium, the pair/tetrad (designated 2/4) cells in a young culture grew and divided but did not separate and became 8-, 16-, and 32-cell units successively. In exponential growth phase, the cells divided in a 16/32 pattern. Potassium ions were equally effective as Na+ in mediating this multicell-formation effect; Mg2+, Li+, and Ca2+ also worked but produced less multiplicity. This effect appears to be species specific. This-section micrographs revealed that in a 16/32-cell unit, eight 2/4 cells were encased in an orderly manner within a large peripheral wall, showing five cycles of septation. Our results suggest the presence of a salt-sensitive mechanism for controlling cell separation in D. radiodurans. Images PMID:2022617

Reprogramming human gallbladder cells into insulin-producing β-like cells

PubMed Central

Benedetti, Eric; Wang, Yuhan; Pelz, Carl; Schug, Jonathan; Kaestner, Klaus H.; Grompe, Markus

2017-01-01

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes. PMID:28813430

Natural killer T cells in health and disease

PubMed Central

Wu, Lan; Van Kaer, Luc

2013-01-01

Natural killer T (NKT) cells are a subset of T lymphocytes that share surface markers and functional characteristics with both conventional T lymphocytes and natural killer cells. Most NKT cells express a semiinvariant T cell receptor that reacts with glycolipid antigens presented by the major histocompatibility complex class I-related protein CD1d on the surface of antigen-presenting cells. NKT cells become activated during a variety of infections and inflammatory conditions, rapidly producing large amounts of immunomodulatory cytokines. NKT cells can influence the activation state and functional properties of multiple other cell types in the immune system and, thus, modulate immune responses against infectious agents, autoantigens, tumors, tissue grafts and allergens. One attractive aspect of NKT cells is that their immunomodulatory activities can be readily harnessed with cognate glycolipid antigens, such as the marine sponge-derived glycosphingolipid alpha-galactosylceramide. These properties of NKT cells are being exploited for therapeutic intervention to prevent or treat cancer, infections, and autoimmune and inflammatory diseases. PMID:21196373

Functional Module Search in Protein Networks based on Semantic Similarity Improves the Analysis of Proteomics Data*

PubMed Central

Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W.; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

2014-01-01

The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system. PMID:24807868

Profiling of lipid and glycogen accumulations under different growth conditions in the sulfothermophilic red alga Galdieria sulphuraria.

PubMed

Sakurai, Toshihiro; Aoki, Motohide; Ju, Xiaohui; Ueda, Tatsuya; Nakamura, Yasunori; Fujiwara, Shoko; Umemura, Tomonari; Tsuzuki, Mikio; Minoda, Ayumi

2016-01-01

The unicellular red alga Galdieria sulphuraria grows efficiently and produces a large amount of biomass in acidic conditions at high temperatures. It has great potential to produce biofuels and other beneficial compounds without becoming contaminated with other organisms. In G. sulphuraria, biomass measurements and glycogen and lipid analyses demonstrated that the amounts and compositions of glycogen and lipids differed when cells were grown under autotrophic, mixotrophic, and heterotrophic conditions. Maximum biomass production was obtained in the mixotrophic culture. High amounts of glycogen were obtained in the mixotrophic cultures, while the amounts of neutral lipids were similar between mixotrophic and heterotrophic cultures. The amounts of neutral lipids were highest in red algae, including thermophiles. Glycogen structure and fatty acids compositions largely depended on the growth conditions. Copyright © 2015. Published by Elsevier Ltd.

Rosa hybrida orcinol O-methyl transferase-mediated production of pterostilbene in metabolically engineered grapevine cell cultures.

PubMed

Martínez-Márquez, Ascensión; Morante-Carriel, Jaime A; Palazon, Javier; Bru-Martínez, Roque

2018-05-25

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties. Copyright © 2018 Elsevier B.V. All rights reserved.

Crucial role of gamma interferon-producing CD4+ Th1 cells but dispensable function of CD8+ T cell, B cell, Th2, and Th17 responses in the control of Brucella melitensis infection in mice.

PubMed

Vitry, Marie-Alice; De Trez, Carl; Goriely, Stanislas; Dumoutier, Laure; Akira, Shizuo; Ryffel, Bernhard; Carlier, Yves; Letesson, Jean-Jacques; Muraille, Eric

2012-12-01

Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4(+) T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8(+) T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.

Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow-Derived Mouse Mesenchymal Stem Cells via a p53 Dependent Mechanism: Implications for Long-Term Culture Expansion

PubMed Central

Boregowda, Siddaraju; Krishnappa, Veena; Chambers, Jeremy; LoGrasso, Phillip V.; Lai, Wen-Tzu; Ortiz, Luis A.; Phinney, Donald G.

2013-01-01

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A and BAX expression and mitochondrial ROS generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immuno-depletion, and supported sustained MSC growth resulting in a 2300-fold increase in cumulative cell yield by 4th passage. MSCs cultured in 5% oxygen also exhibited enhanced tri-lineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA mediated knockdown of p53 in wild type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro. PMID:22367737

Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus).

PubMed

Soler, Carles; Contell, Jesús; Bori, Lorena; Sancho, María; García-Molina, Almudena; Valverde, Anthony; Segarvall, Jan

2017-01-01

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component (PC) analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations: SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed: SP1: large oval cells; SP2: medium size elongated cells; and SP3: small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations - SP1: high oscillatory motility, large and short heads; SP2: medium velocity with small and short heads; SP3: slow motion small and elongated cells; and SP4: high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans.

Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus)

PubMed Central

Soler, Carles; Contell, Jesús; Bori, Lorena; Sancho, María; García-Molina, Almudena; Valverde, Anthony; Segarvall, Jan

2017-01-01

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component (PC) analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations: SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed: SP1: large oval cells; SP2: medium size elongated cells; and SP3: small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations – SP1: high oscillatory motility, large and short heads; SP2: medium velocity with small and short heads; SP3: slow motion small and elongated cells; and SP4: high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans. PMID:27751987

Tumour ischaemia by interferon-γ resembles physiological blood vessel regression.

PubMed

Kammertoens, Thomas; Friese, Christian; Arina, Ainhoa; Idel, Christian; Briesemeister, Dana; Rothe, Michael; Ivanov, Andranik; Szymborska, Anna; Patone, Giannino; Kunz, Severine; Sommermeyer, Daniel; Engels, Boris; Leisegang, Matthias; Textor, Ana; Fehling, Hans Joerg; Fruttiger, Marcus; Lohoff, Michael; Herrmann, Andreas; Yu, Hua; Weichselbaum, Ralph; Uckert, Wolfgang; Hübner, Norbert; Gerhardt, Holger; Beule, Dieter; Schreiber, Hans; Blankenstein, Thomas

2017-05-04

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

Effect of Induced Pluripotent Stem Cell Technology in Blood Banking

PubMed Central

Focosi, Daniele

2016-01-01

Summary Population aging has imposed cost-effective alternatives to blood donations. Artificial blood is still at the preliminary stages of development, and the need for viable cells seems unsurmountable. Because large numbers of viable cells must be promptly available for clinical use, stem cell technologies, expansion, and banking represent ideal tools to ensure a regular supply. Provided key donors can be identified, induced pluripotent stem cell (iPSC) technology could pave the way to a new era in transfusion medicine, just as it is already doing in many other fields of medicine. The present review summarizes the current state of research on iPSC technology in the field of blood banking, highlighting hurdles, and promises. Significance The aging population in Western countries is causing a progressive reduction of blood donors and a constant increase of blood recipients. Because blood is the main therapeutic option to treat acute hemorrhage, cost-effective alternatives to blood donations are being actively investigated. The enormous replication capability of induced pluripotent stem cells and their promising results in many other fields of medicine could be an apt solution to produce the large numbers of viable cells required in transfusion and usher in a new era in transfusion medicine. The present report describes the potentiality, technological hurdles, and promises of induced pluripotent stem cells to generate red blood cells by redifferentiation. PMID:26819256

High Molecular Weight Isoforms of Growth Hormone In Cells of the Immune System

PubMed Central

Weigent, Douglas A.

2013-01-01

A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100 kDa, 65 kDa, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 kDa and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm whereas the lower molecular weight 65 kDa and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost three-fold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress. PMID:21741628

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

Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong

Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None ofmore » the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.« less

Transcranial unifocal stimulation in rabbit: subcutaneous and meningeal changes.

PubMed

Sancesario, G; Massa, R; Petrillo, S; Nottola, S A; Correr, S; Rossini, P M

1989-01-01

The possible acute morphological changes induced by electrical transcranial unifocal stimulation (eTCS) in the rabbit extracerebral tissues were studied by light and scanning electron microscopy. In order to do this, a wide range of electric stimuli with respect to those employed in the clinical practice were utilized. Either surface electrodes were attached to the scalp, or needle electrodes were infixed in the subcutaneous tissue. Beneath the cathode a blood extravasation was constantly observed in the subcutaneous tissue of the scalp; the different electrode arrays produced either a large hemorrhagic lesion or a few petechiae. Beneath the anode, the damage was limited to the scalp, or reached the meninges when stimuli longer than 0.2 ms were used. Irrespective of the electrode arrays, the scalp and the dura mater displayed hemorrhagic petechiae over a limited area about 2-3 mm in extent. Moreover, the leptomeningeal membrane was microscopically disrupted over an area less than 1 mm large; therein the squamous, overlapping cells were transformed into fusiform or macrophage-like cells. Unduly intense eTCS produces evident hemorrhagic lesions in the scalp and in the dura mater, whereas it induces microscopic, reactive changes in the leptomeninx.

Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

NASA Technical Reports Server (NTRS)

Schatten, H.; Lewis, M. L.; Chakrabarti, A.

2001-01-01

The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985. c 2001. Elsevier Science Ltd. All rights reserved.

Single cell assessment of yeast metabolic engineering for enhanced lipid production using Raman and AFM-IR imaging.

PubMed

Kochan, Kamila; Peng, Huadong; Wood, Bayden R; Haritos, Victoria S

2018-01-01

Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. Firstly, a strong correlation ( r 2  > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. Vibrational spectroscopy analysis allowed the simultaneous measurement of strain variability in metabolite production and impact on cellular structures as a result of different gene introductions or knockouts, within a lipid metabolic engineering strategy and these inform the next steps in comprehensive lipid engineering. Additionally, single cell spectroscopic analysis measures heterogeneity in metabolite production across microbial cultures under genetic modification, an emerging issue for efficient biotechnological production.

Large area silicon sheet by EFG

NASA Technical Reports Server (NTRS)

Kalejs, J. P.

1982-01-01

Work carried out on the JPL Flat Plate Solar Array Project, for the purpose of developing a method for silicon ribbon production by Edge-defined Film-fed Growth (EEG) for use as low-cost substrate material in terrestrial solar cell manufacture, is described. A multiple ribbon furnace unit that is designed to operate on a continuous basis for periods of at least one week, with melt replenishment and automatic ribbon width control, and to produce silicon sheet at a rate of one square meter per hour, was constructed. Program milestones set for single ribbon furnace operation to demonstrate basic EEG system capabilities with respect to growth speed, thickness and cell performance were achieved for 10 cm wide ribbon: steady-state growth at 4 cm/min and 200 micron thickness over periods of an hour and longer was made routine, and a small area cell efficiency of 13+% demonstrated. Large area cells of average efficiency of 10 to 11%, with peak values of 11 to 12% were also achieved. The integration of these individual performance levels into multiple ribbon furnace operation was not accomplished.

Interleukin 4: signalling mechanisms and control of T cell differentiation.

PubMed

Paul, W E

1997-01-01

Interleukin 4 (IL-4) is a pleiotropic type I cytokine that controls both growth and differentiation among haemopoietic and non-haemopoietic cells. Its receptor is a heterodimer. One chain, the IL-4R alpha chain, binds IL-4 with high affinity and determines the nature of the biochemical signals that are induced. The second chain, gamma c, is required for the induction of such signals. IL-4-mediated growth depends upon activation events that involve phosphorylation of Y497 of IL-4R alpha, leading to the binding and phosphorylation of 4PS/IRS-2 in haemopoietic cells and of IRS-1 in non-haemopoietic cells. By contrast, IL-4-mediated differentiation events depend upon more distal regions of the IL-4R alpha chain that include a series of STAT-6 binding sites. The distinctive roles of these receptor domains was verified by receptor-reconstruction experiments. The 'growth' and 'differentiation' domains of the IL-4R alpha chain, independently expressed as chimeric structures with a truncated version of the IL-2R beta chain, were shown to convey their functions to the hybrid receptor. The critical role of STAT-6 in IL-4-mediated gene activation and differentiation was made clear by the finding that lymphocytes from STAT-6 knockout mice are strikingly deficient in these functions but have retained the capacity to grow, at least partially, in response to IL-4. IL-4 plays a central role in determining the phenotype of naive CD4+ T cells. In the presence of IL-4, newly primed naive T cells develop into IL-4 producers while in its absence they preferentially become gamma-interferon (IFN-gamma) producers. Recently, a specialized subpopulation of T cells, CD4+/NK1.1+ cells, has been shown to produce large amounts of IL-4 upon stimulation. Two examples of mice with deficiencies in these cells are described--beta 2-microglobulin knockout mice and SJL mice. Both show defects in the development of IL-4-producing cells and in the increase in serum IgE in response to stimulation with the polyclonal stimulant anti-IgD. Both sets of mice have major diminutions in the number of CD4+/ NK1.1+ T cells, strongly indicating an important role of these cells in some but not all IgE responses to physiologic stimuli.

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation

PubMed Central

Santos, Jerran; Milthorpe, Bruce K; Herbert, Benjamin R; Padula, Matthew P

2017-01-01

Background Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Methods Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. Results The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. Conclusion The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs. PMID:28844130

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation.

PubMed

Santos, Jerran; Milthorpe, Bruce K; Herbert, Benjamin R; Padula, Matthew P

2017-11-30

Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

Proliferation zones in the axolotl brain and regeneration of the telencephalon

PubMed Central

2013-01-01

Background Although the brains of lower vertebrates are known to exhibit somewhat limited regeneration after incisional or stab wounds, the Urodele brain exhibits extensive regeneration after massive tissue removal. Discovering whether and how neural progenitor cells that reside in the ventricular zones of Urodeles proliferate to mediate tissue repair in response to injury may produce novel leads for regenerative strategies. Here we show that endogenous neural progenitor cells resident to the ventricular zone of Urodeles spontaneously proliferate, producing progeny that migrate throughout the telencephalon before terminally differentiating into neurons. These progenitor cells appear to be responsible for telencephalon regeneration after tissue removal and their activity may be up-regulated by injury through an olfactory cue. Results There is extensive proliferation of endogenous neural progenitor cells throughout the ventricular zone of the adult axolotl brain. The highest levels are observed in the telencephalon, especially the dorsolateral aspect, and cerebellum. Lower levels are observed in the mesencephalon and rhombencephalon. New cells produced in the ventricular zone migrate laterally, dorsally and ventrally into the surrounding neuronal layer. After migrating from the ventricular zone, the new cells primarily express markers of neuronal differentiative fates. Large-scale telencephalic tissue removal stimulates progenitor cell proliferation in the ventricular zone of the damaged region, followed by proliferation in the tissue that surrounds the healing edges of the wound until the telencephalon has completed regeneration. The proliferative stimulus appears to reside in the olfactory system, because telencephalic regeneration does not occur in the brains of olfactory bulbectomized animals in which the damaged neural tissue simply heals over. Conclusion There is a continual generation of neuronal cells from neural progenitor cells located within the ventricular zone of the axolotl brain. Variable rates of proliferation were detected across brain regions. These neural progenitor cells appear to mediate telencephalic tissue regeneration through an injury-induced olfactory cue. Identification of this cue is our future goal. PMID:23327114

Proliferation zones in the axolotl brain and regeneration of the telencephalon.

PubMed

Maden, Malcolm; Manwell, Laurie A; Ormerod, Brandi K

2013-01-17

Although the brains of lower vertebrates are known to exhibit somewhat limited regeneration after incisional or stab wounds, the Urodele brain exhibits extensive regeneration after massive tissue removal. Discovering whether and how neural progenitor cells that reside in the ventricular zones of Urodeles proliferate to mediate tissue repair in response to injury may produce novel leads for regenerative strategies. Here we show that endogenous neural progenitor cells resident to the ventricular zone of Urodeles spontaneously proliferate, producing progeny that migrate throughout the telencephalon before terminally differentiating into neurons. These progenitor cells appear to be responsible for telencephalon regeneration after tissue removal and their activity may be up-regulated by injury through an olfactory cue. There is extensive proliferation of endogenous neural progenitor cells throughout the ventricular zone of the adult axolotl brain. The highest levels are observed in the telencephalon, especially the dorsolateral aspect, and cerebellum. Lower levels are observed in the mesencephalon and rhombencephalon. New cells produced in the ventricular zone migrate laterally, dorsally and ventrally into the surrounding neuronal layer. After migrating from the ventricular zone, the new cells primarily express markers of neuronal differentiative fates. Large-scale telencephalic tissue removal stimulates progenitor cell proliferation in the ventricular zone of the damaged region, followed by proliferation in the tissue that surrounds the healing edges of the wound until the telencephalon has completed regeneration. The proliferative stimulus appears to reside in the olfactory system, because telencephalic regeneration does not occur in the brains of olfactory bulbectomized animals in which the damaged neural tissue simply heals over. There is a continual generation of neuronal cells from neural progenitor cells located within the ventricular zone of the axolotl brain. Variable rates of proliferation were detected across brain regions. These neural progenitor cells appear to mediate telencephalic tissue regeneration through an injury-induced olfactory cue. Identification of this cue is our future goal.

Improved technique that allows the performance of large-scale SNP genotyping on DNA immobilized by FTA technology.

PubMed

He, Hongbin; Argiro, Laurent; Dessein, Helia; Chevillard, Christophe

2007-01-01

FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. The number of punches that can normally be obtained from a single specimen card are often however, insufficient for the testing of the large numbers of loci required to identify genetic factors that control human susceptibility or resistance to multifactorial diseases. In this study, we propose an improved technique to perform large-scale SNP genotyping. We applied a whole genome amplification method to amplify DNA from buccal cell samples stabilized using FTA technology. The results show that using the improved technique it is possible to perform up to 15,000 genotypes from one buccal cell sample. Furthermore, the procedure is simple. We consider this improved technique to be a promising methods for performing large-scale SNP genotyping because the FTA technology simplifies the collection, shipment, archiving and purification of DNA, while whole genome amplification of FTA card bound DNA produces sufficient material for the determination of thousands of SNP genotypes.

Activation of Meiosis-Specific Genes is Associated with Depolyploidization of Human Tumor Cells Following Radiation-Induced Mitotic Catastrophe

PubMed Central

Ianzini, Fiorenza; Kosmacek, Elizabeth A.; Nelson, Elke S.; Napoli, Eleonora; Erenpreisa, Jekaterina; Kalejs, Martins; Mackey, Michael A.

2009-01-01

Cancer is frequently characterized histologically by the appearance of large cells that are either aneuploid or polyploid. Aneuploidy and polyploidy are hallmarks of radiation-induced mitotic catastrophe (MC), a common phenomenon occurring in tumor cells with impaired p53 function exposed to various cytotoxic and genotoxic agents. MC is characterized by altered expression of mitotic regulators, untimely and abnormal cell division, delayed DNA damage, and changes in morphology. We report here that cells undergoing radiation-induced MC are more plastic with regards to ploidy and that this plasticity allows them to reorganize their genetic material through reduction divisions to produce smaller cells morphologically indistinguishable from control cells. Experiments conducted with the Large Scale Digital Cell Analysis System (LSDCAS) are discussed that show that a small fraction of polyploid cancer cells formed via radiation-induced MC can survive and start a process of depolyploidization that yields various outcomes. While most multipolar divisions failed and cell fusion occurred; some of these divisions were successful and originated a variety of cell progeny characterized by different ploidy. Among these ploidy phenotypes, a progeny of small mononucleated cells, indistinguishable from the untreated control cells, is often seen. We report here evidence that meiosis-specific genes are expressed in the polyploid cells during depolyploidization. Tumor cells might take advantage of the temporary change from a pro-mitotic to a pro-meiotic division regimen to facilitate depolyploidization and restore the proliferative state of the tumor cell population. These events might be mechanisms by which tumor progression and resistance to treatment occur in vivo. PMID:19258501

Changes in E-cadherin rigidity sensing regulate cell adhesion

PubMed Central

Collins, Caitlin; Pruitt, Beth L.; Nelson, W. James

2017-01-01

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion. PMID:28674019

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis.

PubMed

Yoshida, Shosei

2012-09-01

Spermatogenesis in mice and other mammalians is supported by a robust stem cell system. Stem cells maintain themselves and continue to produce progeny that will differentiate into sperm over a long period. The pioneering studies conducted from the 1950s to the 1970s, which were based largely on extensive morphological analyses, have established the fundamentals of mammalian spermatogenesis and its stem cells. The prevailing so-called A(single) (A(s)) model, which was originally established in 1971, proposes that singly isolated A(s) spermatogonia are in fact the stem cells. In 1994, the first functional stem cell assay was established based on the formation of repopulating colonies after transplantation in germ cell-depleted host testes, which substantially accelerated the understanding of spermatogenic stem cells. However, because testicular tissues are dissociated into single-cell suspension before transplantation, it was impossible to evaluate the A(s) and other classical models solely by this technique. From 2007 onwards, functional assessment of stem cells without destroying the tissue architecture has become feasible by means of pulse-labeling and live-imaging strategies. Results obtained from these experiments have been challenging the classical thought of stem cells, in which stem cells are a limited number of specialized cells undergoing asymmetric division to produce one self-renewing and one differentiating daughter cells. In contrast, the emerging data suggest that an extended and heterogeneous population of cells exhibiting different degrees of self-renewing and differentiating probabilities forms a reversible, flexible, and stochastic stem cell system as a population. These features may lead to establishment of a more universal principle on stem cells that is shared by other systems.

The excretory-secretory products of Echinococcus granulosus protoscoleces directly regulate the differentiation of B10, B17 and Th17 cells.

PubMed

Pan, Wei; Hao, Wen-Ting; Shen, Yu-Juan; Li, Xiang-Yang; Wang, Yan-Juan; Sun, Fen-Fen; Yin, Jian-Hai; Zhang, Jing; Tang, Ren-Xian; Cao, Jian-Ping; Zheng, Kui-Yang

2017-07-21

Excretory-secretory products (ESPs) released by helminths are well-known to regulate T cell responses in the host. However, their direct influence in the differentiation of naïve T cells, and especially B cells, remains largely unknown. This study investigated the effects of Echinococcus granulosus protoscoleces ESPs (EgPSC-ESPs) on the differentiation of IL-10-producing B cells (B10), IL-17A-producing B cells (B17) and Th17 cells. BALB/c mice injected with EgPSC were used to evaluate the in vivo profiles of B10, B17 and Th17 cells. In vitro purified CD19 + B and naïve CD4 + T cells were cultured in the presence of native, heat-inactivated or periodate-treated EgPSC-ESPs, and the differentiation of these cell subsets were compared. In contrast to the control group, infected mice showed higher frequencies of B10, B17 and Th17 cells, and higher levels of IL-10 and IL-17A in the sera. Interestingly, B17 cells were first identified to express CD19 + CD1d high . In vitro, B cells cultured with native ESPs exhibited a higher percentage of B10 cells but lower percentage of B17 and Th17 cells compared to the PBS group. Moreover, the relative expression of IL-10 and IL-17A mRNA were consistent with the altered frequencies. However, ESPs subjected to heat-inactivation or periodate treatment exhibited an inverse effect on the induction of these cell subsets. Our findings indicate that ESPs released by EgPSC can directly regulate the differentiation of B10, B17 and Th17 cells, which appear to be heat-labile and carbohydrate-dependent.

Engineering cells for cell culture bioprocessing--physiological fundamentals.

PubMed

Seth, Gargi; Hossler, Patrick; Yee, Joon Chong; Hu, Wei-Shou

2006-01-01

In the past decade, we have witnessed a tremendous increase in the number of mammalian cell-derived therapeutic proteins with clinical applications. The success of making these life-saving biologics available to the public is partly due to engineering efforts to enhance process efficiency. To further improve productivity, much effort has been devoted to developing metabolically engineered producing cells, which possess characteristics favorable for large-scale bioprocessing. In this article we discuss the fundamental physiological basis for cell engineering. Different facets of cellular mechanisms, including metabolism, protein processing, and the balancing pathways of cell growth and apoptosis, contribute to the complex traits of favorable growth and production characteristics. We present our assessment of the current state of the art by surveying efforts that have already been undertaken in engineering cells for a more robust process. The concept of physiological homeostasis as a key determinant and its implications on cell engineering is emphasized. Integrating the physiological perspective with cell culture engineering will facilitate attainment of dream cells with superlative characteristics.

Microspheres

NASA Technical Reports Server (NTRS)

1990-01-01

Vital information on a person's physical condition can be obtained by identifying and counting the population of T-cells and B-cells, lymphocytes of the same shape and size that help the immune system protect the body from the invasion of disease. The late Dr. Alan Rembaum developed a method for identifying the cells. The method involved tagging the T-cells and B-cells with microspheres of different fluorescent color. Microspheres, which have fluorescent dye embedded in them, are chemically treated so that they can link with antibodies. With the help of a complex antibody/antigen reaction, the microspheres bind themselves to specific 'targets,' in this case the T-cells or B-cells. Each group of cells can then be analyzed by a photoelectronic instrument at different wavelengths emitted by the fluorescent dyes. Same concept was applied to the separation of cancer cells from normal cells. Microspheres were also used to conduct many other research projects. Under a patent license Magsphere, Inc. is producing a wide spectrum of microspheres on a large scale and selling them worldwide for various applications.

A simple and low-cost platform technology for producing pexiganan antimicrobial peptide in E. coli.

PubMed

Zhao, Chun-Xia; Dwyer, Mirjana Dimitrijev; Yu, Alice Lei; Wu, Yang; Fang, Sheng; Middelberg, Anton P J

2015-05-01

Antimicrobial peptides, as a new class of antibiotics, have generated tremendous interest as potential alternatives to classical antibiotics. However, the large-scale production of antimicrobial peptides remains a significant challenge. This paper reports a simple and low-cost chromatography-free platform technology for producing antimicrobial peptides in Escherichia coli (E. coli). A fusion protein comprising a variant of the helical biosurfactant protein DAMP4 and the known antimicrobial peptide pexiganan is designed by joining the two polypeptides, at the DNA level, via an acid-sensitive cleavage site. The resulting DAMP4(var)-pexiganan fusion protein expresses at high level and solubility in recombinant E. coli, and a simple heat-purification method was applied to disrupt cells and deliver high-purity DAMP4(var)-pexiganan protein. Simple acid cleavage successfully separated the DAMP4 variant protein and the antimicrobial peptide. Antimicrobial activity tests confirmed that the bio-produced antimicrobial peptide has the same antimicrobial activity as the equivalent product made by conventional chemical peptide synthesis. This simple and low-cost platform technology can be easily adapted to produce other valuable peptide products, and opens a new manufacturing approach for producing antimicrobial peptides at large scale using the tools and approaches of biochemical engineering. © 2014 Wiley Periodicals, Inc.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

PubMed

Beer, Lara-Antonia; Tatge, Helma; Schneider, Carmen; Ruschig, Maximilian; Hust, Michael; Barton, Jessica; Thiemann, Stefan; Fühner, Viola; Russo, Giulio; Gerhard, Ralf

2018-06-01

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum , the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile . All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N -terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Hypocrea lixii, novel endophytic fungi producing anticancer agent cajanol, isolated from pigeon pea (Cajanus cajan [L.] Millsp.).

PubMed

Zhao, J; Li, C; Wang, W; Zhao, C; Luo, M; Mu, F; Fu, Y; Zu, Y; Yao, M

2013-07-01

The aim was to isolate, identify and characterize endophytes from pigeon pea (Cajanus cajan [L.] Millsp.), as novel producer of cajanol and its in vitro cytotoxicity assay. Isolation, identification and characterization of novel endophytes producing cajanol from the roots of pigeon pea were investigated. The endophytes were identified as Hypocrea lixii by morphological and molecular methods. Cajanol produced by endophytes were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). R-18 produced the highest levels of cajanol (322·4 ± 10·6 μg l(-1) or 102·8 ± 6·9 μg g(-1) dry weight of mycelium) after incubation for 7 days. The cytotoxicity towards human lung carcinoma cells (A549) of fungal cajanol was investigated in vitro. First, a novel endophyte Hypocrea lixii, producing anticancer agent cajanol, was isolated from the host pigeon pea (Cajanus cajan [L.] Millsp.). Fungal cajanol possessed stronger cytotoxicity activity towards A549 cells in time- and dose-dependent manners. This endophyte is a potential handle for scientific and commercial exploitation, and it could provide a promising alterative approach for large-scale production of cajanol to satisfy new anticancer drug development. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

Leishmania major large RAB GTPase is highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis.

PubMed

Chamakh-Ayari, Rym; Chenik, Mehdi; Chakroun, Ahmed Sahbi; Bahi-Jaber, Narges; Aoun, Karim; Meddeb-Garnaoui, Amel

2017-04-17

We previously identified a Leishmania (L.) major large RAB GTPase (LmlRAB), a new atypical RAB GTPase protein. It is highly conserved in Leishmania species while displaying low level of homology with mammalian homologues. Leishmania small RAB GTPases proteins have been involved in regulation of exocytic and endocytic pathways whereas the role of large RAB GTPases proteins has not been characterized yet. We report here the immunogenicity of both recombinant rLmlRAB and rLmlRABC, in individuals with immunity against L. major or L. infantum. PBMC were isolated from individuals cured of L. major (CCLm) or from healthy individuals. The latter were subdivided into high or low IFN-γ responders. Healthy high IFN-γ responders, considered as asymptomatics, were living in an endemic area for L. major (HHRLm) or L. infantum (HHRLi). Healthy low IFN-γ responders (HLR) were considered as naïve controls. Cells from all volunteers were stimulated with rLmlRAB or rLmlRABC. Cytokines were analysed by CBA and ELISA and phenotypes of IFN-γ-producing cells were analysed by flow cytometry. Both rLmlRAB and rLmlRABC induced high significant levels of IFN-γ in CCLm, HHRLm and HHRLi groups. Phenotype analysis of rLmlRAB and rLmlRABC-stimulated T cells in CCLm individuals showed a significant increase in the percentage of specific IFN-γ-producing CD4+ and CD8+ T cells. rLmlRAB induced significant granzyme B levels in CCLm and HHRLm. Low but significant granzyme B levels were detected in naïve group. IL-10 was detected in immune and naïve individuals. We showed that rLmlRAB protein and its divergent carboxy-terminal part induced a predominant Th1 response in individuals immune to L. major or L. infantum. Our results suggest that rLmlRAB and rLmlRABC proteins are potential cross-species vaccine candidates against cutaneous and visceral leishmaniasis.

Effect of a transient period of ischemia on myocardial cells. II. Fine structure during the first few minutes of reflow.

PubMed

Kloner, R A; Ganote, C E; Whalen, D A; Jennings, R B

1974-03-01

Changes produced in the posterior papillary muscle of the dog following 40 minutes of circumflex artery occlusion and 0 to 20 minutes of blood reflow were studied by electron miroscopy. With no reflow of blood, myocardial cells were modestly swollen, contained amorphous matrix densities in the mitochondria, had aggregation and margination of nuclear chromatin and relaxation of myofibrils. With as little as 2 minutes of blood reflow, cells developed contraction bands and were greatly swollen due to a generalized increase in sarcoplasmic space, formation of vacuoles and swelling of mitochondria. Frequently, cell membranes were lifted away from the myofibers, forming large subsarcolemmal blebs which appeared capable of compressing adjacent capillaries. The extracellular space did not appear to be enlarged, and the marked tissue edema found after reflow was due primarily to accumulation of intracellular fluid. In addition to explosive cell swelling, there was, over the 2- to 20-minute period of reflow, a progressive increase in size and number of granular mitochondrial dense bodies of the calcium accumulation type. No significant changes in lysosomes were observed. The speed with which the morphologic changes developed during very early reflow periods suggests that irreversible ischemic injury produces a defect in cell volume regulation during the phase of ischemia and that this defect becomes manifest if arterial flow is restored to the affected cells.

Human Macrophages Escape Inhibition of Major Histocompatibility Complex-Dependent Antigen Presentation by Cytomegalovirus and Drive Proliferation and Activation of Memory CD4+ and CD8+ T Cells.

PubMed

Frascaroli, Giada; Lecher, Carina; Varani, Stefania; Setz, Corinna; van der Merwe, Johannes; Brune, Wolfram; Mertens, Thomas

2018-01-01

Human cytomegalovirus (HCMV) persistently infects 40-90% of the human population but in the face of a normal immune system, viral spread and dissemination are efficiently controlled thus preventing clinically signs and disease. HCMV-infected hosts produce a remarkably large amount of HCMV-specific CD4 + and CD8 + T cells that can even reach 20-50% of total T memory cells in the elderly. How HCMV may elicit such large and long-lasting T-cell responses in the absence of detectable viremia has not been elucidated yet. Additionally, HCMV is known to encode several gene products that potently inhibit T-cell recognition of infected cells. The best characterized are the four immune evasive US2, US3, US6, and US11 genes that by different mechanisms account for major histocompatibility complex (MHC) class I and class II degradation and intracellular retention in infected cells. By infecting M1 and M2 human macrophages (Mφ) with the wild-type HCMV strain TB40E or a mutant virus deleted of the four immune evasive genes US2, US3, US6, and US11, we demonstrated that human Mφ counteract the inhibitory potential of the US2-11 genes and remain capable to present peptides via MHC class I and class II molecules. Moreover, by sorting the infected and bystander cells, we provide evidence that both infected and bystander Mφ contribute to antigen presentation to CD4 + and CD8 + T cells. The T cells responding to TB40E-infected Mφ show markers of the T effector memory compartment, produce interferon-γ, and express the lytic granule marker CD107a on the cell surface, thus mirroring the HCMV-specific T cells present in healthy seropositive individuals. All together, our findings reveal that human Mφ escape inhibition of MHC-dependent antigen presentation by HCMV and continue to support T cell proliferation and activation after HCMV infection. Taking into account that Mφ are natural targets of HCMV infection and a site of viral reactivation from latency, our findings support the hypothesis that Mφ play crucial roles for the lifelong maintenance and expansion of HCMV-committed T cells in the human host.

Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.

PubMed

Tauchman, Eric C; Boehm, Frederick J; DeLuca, Jennifer G

2015-12-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore-microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore-microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.

Fabrication of large size alginate beads for three-dimensional cell-cluster culture

NASA Astrophysics Data System (ADS)

Zhang, Zhengtao; Ruan, Meilin; Liu, Hongni; Cao, Yiping; He, Rongxiang

2017-08-01

We fabricated large size alginate beads using a simple microfluidic device under a co-axial injection regime. This device was made by PDMS casting with a mold formed by small diameter metal and polytetrafluorothylene tubes. Droplets of 2% sodium alginate were generated in soybean oil through the device and then cross-linked in a 2% CaCl2 solution, which was mixed tween80 with at a concentration of 0.4 to 40% (w/v). Our results showed that the morphology of the produced alginate beads strongly depends on the tween80 concentration. With the increase of concentration of tween80, the shape of the alginate beads varied from semi-spherical to tailed-spherical, due to the decrease of interface tension between oil and cross-link solution. To access the biocompatibility of the approach, MCF-7 cells were cultured with the alginate beads, showing the formation of cancer cells clusters which might be useful for future studies.

Short-Range Temporal Interactions in Sleep; Hippocampal Spike Avalanches Support a Large Milieu of Sequential Activity Including Replay

PubMed Central

Mahoney, J. Matthew; Titiz, Ali S.; Hernan, Amanda E.; Scott, Rod C.

2016-01-01

Hippocampal neural systems consolidate multiple complex behaviors into memory. However, the temporal structure of neural firing supporting complex memory consolidation is unknown. Replay of hippocampal place cells during sleep supports the view that a simple repetitive behavior modifies sleep firing dynamics, but does not explain how multiple episodes could be integrated into associative networks for recollection during future cognition. Here we decode sequential firing structure within spike avalanches of all pyramidal cells recorded in sleeping rats after running in a circular track. We find that short sequences that combine into multiple long sequences capture the majority of the sequential structure during sleep, including replay of hippocampal place cells. The ensemble, however, is not optimized for maximally producing the behavior-enriched episode. Thus behavioral programming of sequential correlations occurs at the level of short-range interactions, not whole behavioral sequences and these short sequences are assembled into a large and complex milieu that could support complex memory consolidation. PMID:26866597

Stable kinetochore–microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells

PubMed Central

Tauchman, Eric C.; Boehm, Frederick J.; DeLuca, Jennifer G.

2015-01-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. PMID:26620470

Collapsing Aged Culture of the Cyanobacterium Synechococcus elongatus Produces Compound(s) Toxic to Photosynthetic Organisms

PubMed Central

Cohen, Assaf; Sendersky, Eleonora; Carmeli, Shmuel; Schwarz, Rakefet

2014-01-01

Phytoplankton mortality allows effective nutrient cycling, and thus plays a pivotal role in driving biogeochemical cycles. A growing body of literature demonstrates the involvement of regulated death programs in the abrupt collapse of phytoplankton populations, and particularly implicates processes that exhibit characteristics of metazoan programmed cell death. Here, we report that the cell-free, extracellular fluid (conditioned medium) of a collapsing aged culture of the cyanobacterium Synechococcus elongatus is toxic to exponentially growing cells of this cyanobacterium, as well as to a large variety of photosynthetic organisms, but not to eubacteria. The toxic effect, which is light-dependent, involves oxidative stress, as suggested by damage alleviation by antioxidants, and the very high sensitivity of a catalase-mutant to the conditioned medium. At relatively high cell densities, S. elongatus cells survived the deleterious effect of conditioned medium in a process that required de novo protein synthesis. Application of conditioned medium from a collapsing culture caused severe pigment bleaching not only in S. elongatus cells, but also resulted in bleaching of pigments in a cell free extract. The latter observation indicates that the elicited damage is a direct effect that does not require an intact cell, and therefore, is mechanistically different from the metazoan-like programmed cell death described for phytoplankton. We suggest that S. elongatus in aged cultures are triggered to produce a toxic compound, and thus, this process may be envisaged as a novel regulated death program. PMID:24959874

Photo-recovery of electron-irradiated GaAs solar cells

NASA Technical Reports Server (NTRS)

Meulenberg, Andrew

1995-01-01

The first long-term (3000 hours) UV testing of unirradiated and 1 MeV electron-irradiated GaAs solar cells, with multilayer-coated coverslides to reduce solar array operating temperature, has produced some unexpected and important results. Two results, independent of the coverslide coatings, are of particular importance in terms of the predictability of GaAs solar-array lifetime in space: ( 1) The GaAs/Ge solar cells used for this series of tests displayed a much higher radiation degradation than that predicted based on JPL Solar Cell Radiation Handbook data. Covered cells degraded more in Isc than did bare cells. Short-term illumination at 60 C did not produce significant recovery (-1%) of the radiation damage. (2) However, electron radiation damage to these GaAs solar celIs anneals at 40 C when exposed to approximately 1 sun AM0 UV light sources for extended periods. The effect appears to be roughly linear with time (-1% of lsc per 1000 UVSH), is large (greater than or equal to 3%), and has not yet saturated (at 3000 hours). This photo-recovery of radiation damage to GaAs solar cells is a new effect and potentially important to the spacecraft community. The figure compares the effects of extended UV on irradiated and unirradiated GaAs solar cells with INTELSAT-6 Si cells. The effect and its generality, the extent of and conditions for photo-recovery, and the implications of such recovery for missions in radiation environments have not yet been determined.

Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

NASA Astrophysics Data System (ADS)

Salick, Max R.

The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

Reciprocity between Regulatory T Cells and Th17 Cells: Relevance to Polarized Immunity in Leprosy

PubMed Central

Sadhu, Soumi; Khaitan, Binod Kumar; Joshi, Beenu; Sengupta, Utpal; Nautiyal, Arvind Kumar; Mitra, Dipendra Kumar

2016-01-01

T cell defect is a common feature in lepromatous or borderline lepromatous leprosy (LL/BL) patients in contrast to tuberculoid or borderline tuberculoid type (TT/BT) patients. Tuberculoid leprosy is characterized by strong Th1-type cell response with localized lesions whereas lepromatous leprosy is hallmarked by its selective Mycobacterium leprae specific T cell anergy leading to disseminated and progressive disease. FoxP3+ Regulatory T cells (Treg) which are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases also dampen proinflammatory T cells that include T helper 17 (Th17) cells. This study is aimed at evaluating the role of Treg cells in influencing other effector T cells and its relationship with the cytokine polarized state in leprosy patients. Peripheral blood mononuclear cells from of BT/TT (n = 15) and BL/LL (n = 15) patients were stimulated with M. leprae antigen (WCL) in presence of golgi transport inhibitor monensin for FACS based intracellular cytokine estimation. The frequency of Treg cells showed >5-fold increase in BL/LL in comparison to BT/TT and healthy contacts. These cells produced suppressive cytokine, IL-10 in BL/LL as opposed to BT/TT (p = 0.0200) indicating their suppressive function. The frequency of Th17 cells (CD4, CD45RO, IL-17) was, however, higher in BT/TT. Significant negative correlation (r = -0.68, P = 0.03) was also found between IL-10 of Treg cells and IL-17+ T cells in BL/LL. Blocking IL-10/TGF-β restored the IL-17+ T cells in BL/LL patients. Simultaneously, presence of Th17 related cytokines (TGF-β, IL-6, IL-17 and IL-23) decreased the number of FoxP3+ Treg cells concomitantly increasing IL-17 producing CD4+ cells in lepromatous leprosy. Higher frequency of Programmed Death-1/PD-1+ Treg cells and its ligand, PDL-1 in antigen presenting cells (APCs) was found in BL/LL patients. Inhibition of this pathway led to rescue of IFN-γ and IL-17 producing T cells. Results indicate that Treg cells are largely responsible for the kind of immunosuppression observed in BL/LL patients. This study also proves that Treg cells are profoundly affected by the cytokine milieu and this property may be utilized for benefit of the host. PMID:26751584

A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting

PubMed Central

Lindeman, Geoffrey J.; Dagnino, Lina; Gaubatz, Stefan; Xu, Yuhui; Bronson, Roderick T.; Warren, Henry B.; Livingston, David M.

1998-01-01

Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue. PMID:9553039

Vector-averaged gravity alters myocyte and neuron properties in cell culture

NASA Technical Reports Server (NTRS)

Gruener, Raphael; Hoeger, Glenn

1991-01-01

The effect of changes in the gravitational field of developing neurons and myocytes on the development of these cells was investigated using observations of rotated cultures of embryonic spinal neurons and myocytes in a horizontal clinostat, in which rotation produces, from the cells' perspective, a 'vector-free' gravity environment by continous averaging of the vector, thus simulating the microgravity of space. It was found that, at rotation rates between 1 and 50 rpm, cellular and nuclear areas of myocytes become significantly enlarged and the number of presumptive nucleoli increase; in neurons, frequent and large swellings appeared along neuritic shafts. Some of these changes were reversible after the cessation of rotation.

Interleukin-6, Produced by Resident Cells of the Central Nervous System and Infiltrating Cells, Contributes to the Development of Seizures following Viral Infection▿

PubMed Central

Libbey, Jane E.; Kennett, Nikki J.; Wilcox, Karen S.; White, H. Steve; Fujinami, Robert S.

2011-01-01

Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development. PMID:21543484

Cell differentiation: therapeutical challenges in diabetes.

PubMed

Roche, Enrique; Vicente-Salar, Nestor; Arribas, Maribel; Paredes, Beatriz

2012-01-01

Stem cells, derived from either embryonic or adult tissues, are considered to be potential sources of insulin-secreting cells to be transplanted into type 1 and advanced stages of type 2 diabetic patients. Many laboratories have considered this possibility, resulting in a large amount of published protocols, with a wide degree of complexity among them. Our group was the first to report that it was possible to obtain insulin-secreting cells from mouse embryonic stem cells, proving the feasibility of this new challenge. The same observation was immediately reported using human embryonic stem cells. However, the resulting cell product was not properly characterised, affecting the reproducibility of the protocol by other groups. A more elaborated protocol was developed by Lumelsky and co-workers, demonstrating that neuroectodermal cells could be an alternative source for insulin-producing cells. However, the resulting cells of this protocol produced low amounts of the hormone. This aimed other groups to perform key changes in order to improve the insulin content of the resulting cells. Recently, Baetge's group has published a new protocol based on the knowledge accumulated in pancreatic development. In this protocol, human embryonic stem cells were differentiated into islet-like structures through a five step protocol, emulating the key steps during embryonic development of the endocrine pancreas. The final cell product, however, seemed to be in an immature state, thus further improvement is required. Despite this drawback, the protocol represents the culmination of work performed by different groups and offers new research challenges for the investigators in this exciting field. Concerning adult stem cells, the possibility of identifying pancreatic precursors or of reprogramming extrapancreatic derived cells are key possibilities that may circumvent the problems that appear when using embryonic stem cells, such as immune rejection and tumour formation.

Rapid large area fabrication of multiscale through-hole membranes.

PubMed

Tahk, Dongha; Paik, Sang-Min; Lim, Jungeun; Bang, Seokyoung; Oh, Soojung; Ryu, Hyunryul; Jeon, Noo Li

2017-05-16

There are many proposed mechanisms by which single cells can be trapped; among them is the through-hole membrane for the characterization of individual microorganisms. Due to the small scale of the fabricated pores, the construction of through-hole membranes on a large scale and with relatively large areas faces many difficulties. This paper describes novel fabrication methods for a large-area, freestanding micro/nano through-hole membrane constructed from versatile membrane materials using through-hole membranes on a microfluidic chip (THMMC). This process can rapidly (<20 min) fabricate membranes with high fidelity multiscale hole size without residual layers. The through-hole site was easily customizable from the micro to the nanoscale, with a low or high aspect ratio giving rise to reliable membranes. Also, the rigidity and biocompatibility of the through-hole membrane are easily tunable by simple injection of versatile membrane materials to obtain a large area (up to 3600 mm 2 ). Membranes produced in this manner were then applied as a proof of concept for the isolation, cultivation, and quantification of individual micro-algal cells for selection with respect to the growth rate, while controlling the quorum sensing mediated metabolic and proliferative changes.

Impaired plasmacytoid dendritic cell (PDC)-NK cell activity in viremic human immunodeficiency virus infection attributable to impairments in both PDC and NK cell function.

PubMed

Conry, Sara J; Milkovich, Kimberly A; Yonkers, Nicole L; Rodriguez, Benigno; Bernstein, Helene B; Asaad, Robert; Heinzel, Frederick P; Tary-Lehmann, Magdalena; Lederman, Michael M; Anthony, Donald D

2009-11-01

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections impair plasmacytoid dendritic cell (PDC) and natural killer (NK) cell subset numbers and functions, though little is known about PDC-NK cell interactions during these infections. We evaluated PDC-dependent NK cell killing and gamma interferon (IFN-gamma) and granzyme B production, using peripheral blood mononuclear cell (PBMC)-based and purified cell assays of samples from HCV- and HIV-infected subjects. CpG-enhanced PBMC killing and IFN-gamma and granzyme B activity (dependent on PDC and NK cells) were impaired in viremic HIV infection. In purified PDC-NK cell culture experiments, CpG-enhanced, PDC-dependent NK cell activity was cell contact and IFN-alpha dependent, and this activity was impaired in viremic HIV infection but not in HCV infection. In heterologous PDC-NK cell assays, impaired PDC-NK cell killing activity was largely attributable to an NK cell defect, while impaired PDC-NK cell IFN-gamma-producing activity was attributable to both PDC and NK cell defects. Additionally, the response of NK cells to direct IFN-alpha stimulation was defective in viremic HIV infection, and this defect was not attributable to diminished IFN-alpha receptor expression, though IFN-alpha receptor and NKP30 expression was closely associated with killer activity in viremic HIV infection but not in healthy controls. These data indicate that during uncontrolled HIV infection, PDC-dependent NK cell function is impaired, which is in large part attributable to defective IFN-alpha-induced NK cell activity and not to altered IFN-alpha receptor, NKP30, NKP44, NKP46, or NKG2D expression.

Long-term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor

PubMed Central

Kuzin, Igor; Sun, Hongliang; Moshkani, Safiekhatoon; Feng, Changyong; Mantalaris, Athanasios; Wu, JH David; Bottaro, Andrea

2011-01-01

Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g. T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three-dimensional bioreactor system for long-term, functional culture of human bone marrow cells on macroporous microspheres in a packed-bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell-enriched areas. Strikingly, these cultures generated a significant number of antibody-producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three-dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo. PMID:21309085

Long-term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor.

PubMed

Kuzin, Igor; Sun, Hongliang; Moshkani, Safiekhatoon; Feng, Changyong; Mantalaris, Athanasios; Wu, J H David; Bottaro, Andrea

2011-06-01

Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g., T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological, and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three-dimensional bioreactor system for long-term, functional culture of human bone marrow cells on macroporous microspheres in a packed-bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell-enriched areas. Strikingly, these cultures generated a significant number of antibody-producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three-dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo. Copyright © 2011 Wiley Periodicals, Inc.

Development of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular effects.

PubMed

Oostingh, Gertie J; Papaioannou, Eleni; Chasapidis, Leonidas; Akritidis, Theofylaktos; Konstandopoulos, Athanasios G; Duschl, Albert

2013-09-01

Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful than the exposure to a larger number of micrometer-sized particles. An in vitro exposure system was established to expose human alveolar epithelial cells to freshly generated exhaust. Computer simulations were used to determine the optimal flow characteristics and ensure equal exposure conditions for each well of a 6-well plate. A selective particle size sampler was used to continuously deliver diesel soot particles with different particle size distributions to cells in culture. To determine, whether the system could be used for cellular assays, alterations in cytokine production and cell viability of human alveolar A549 cells were determined after 3h on-line exposure followed by a 21-h conventional incubation period. Data indicated that complete diesel engine emission slightly affected pre-stimulated cells, but naive cells were not affected. The fractions containing large or small particles never affected the cells. The experimental set-up allowed a reliable exposure of the cells to the complete exhaust fraction or to the fractions containing either large or small diesel engine emission particles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mesenchymal stem cells: Emerging mechanisms of immunomodulation and therapy

PubMed Central

Glenn, Justin D; Whartenby, Katharine A

2014-01-01

Mesenchymal stem cells (MSCs) are a pleiotropic population of cells that are self-renewing and capable of differentiating into canonical cells of the mesenchyme, including adipocytes, chondrocytes, and osteocytes. They employ multi-faceted approaches to maintain bone marrow niche homeostasis and promote wound healing during injury. Biomedical research has long sought to exploit their pleiotropic properties as a basis for cell therapy for a variety of diseases and to facilitate hematopoietic stem cell establishment and stromal reconstruction in bone marrow transplantation. Early results demonstrated their usage as safe, and there was little host response to these cells. The discovery of their immunosuppressive functions ushered in a new interest in MSCs as a promising therapeutic tool to suppress inflammation and down-regulate pathogenic immune responses in graft-versus-host and autoimmune diseases such as multiple sclerosis, autoimmune diabetes, and rheumatoid arthritis. MSCs produce a large number of soluble and membrane-bound factors, some of which inhibit immune responses. However, the full range of MSC-mediated immune-modulation remains incompletely understood, as emerging reports also reveal that MSCs can adopt an immunogenic phenotype, stimulate immune cells, and yield seemingly contradictory results in experimental animal models of inflammatory disease. The present review describes the large body of literature that has been accumulated on the fascinating biology of MSCs and their complex effects on immune responses. PMID:25426250

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid.

PubMed

Yoneyama, Tohru; Miyata, Keita; Chikai, Tomoyuki; Mikami, Akifumi; Suzuki, Tomonori; Hasegawa, Kimiko; Ikeda, Toshihiko; Watanabe, Toshihiro; Ohyama, Tohru; Niwa, Koichi

2008-12-01

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

Ca2+ mobilization during cell death induction by sodium 5, 6-benzylidene-L-ascorbate.

PubMed

Takahashi, H; Sakagami, H; Ohata, H; Iida, M; Momose, K; Yamamura, M; Takeda, M

1996-01-01

The cytotoxic activity of sodium 5,6-benzylidene-L-ascorbate (SBA) against human KG-1-C glioma and T98G glioblastoma cell lines was augmented by pretreatment of the cells with L-buthionine-[S, R]-sulfoximine (BSO), which reduced the intracellular glutathione concentrations. SBA produced shrunken cells and large DNA fragments, without the induction of nuclear and internucleosomal DNA fragmentation. The rapid elevation of intracellular free Ca2+ concentration observed after SBA treatment was further augmented by BSO pretreatment. A confocal experiment with Fluo-3 fluorescence revealed that SBA markedly elevated the free Ca2+ concentration in the nuclear region, but did not significantly affect that in the cytoplasmic region. The present study suggests that the nuclear accumulation of Ca2+ is an important initial step for cell death induction by SBA.

Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice.

PubMed Central

Zhou, X; Paulsson, G; Stemme, S; Hansson, G K

1998-01-01

Atherosclerosis is an inflammatory-fibrotic response to accumulation of cholesterol in the artery wall. In hypercholesterolemia, low density lipoproteins (LDL) accumulate and are oxidized to proinflammatory compounds in the arterial intima, leading to activation of endothelial cells, macrophages, and T lymphocytes. We have studied immune cell activation and the autoimmune response to oxidized LDL in atherosclerotic apo E-knockout mice. Autoantibodies to oxidized LDL exhibited subclass specificities indicative of T cell help, and the increase in antibody titers in peripheral blood was associated with increased numbers of cytokine-expressing T cells in the spleen. In addition to T cell-dependent antibodies, IgM antibodies to oxidized LDL were also increased in apo E-knockout mice. This suggests that both T cell-dependent and T cell-independent epitopes may be present on oxidized LDL. In moderate hypercholesterolemia, IgG antibodies were largely of the IgG2a isotype, suggesting that T cell help was provided by proinflammatory T helper (Th) 1 cells, which are prominent components of atherosclerotic lesions. In severe hypercholesterolemia induced by cholesterol feeding of apo E-knockout mice, a switch to Th2-dependent help was evident. It was associated with a loss of IFN-gamma-producing Th1 cells in the spleen, whereas IL-4-producing Th2 cells were more resistant to hypercholesterolemia. IFN-gamma but not IL-4 mRNA was detected in atherosclerotic lesions of moderately hypercholesterolemic apo E-knockout mice, but IL-4 mRNA appeared in the lesions when mice were made severely hypercholesterolemic by cholesterol feeding. These data show that IFN-gamma-producing Th1 cells infiltrate atherosclerotic lesions and provide T cell help for autoimmune responses to oxidized LDL in apo E-knockout mice. However, severe hypercholesterolemia is associated with a switch from Th1 to Th2, which results not only in the formation of IgG1 autoantibodies to oxidized LDL, but also in the appearance of Th2-type cytokines in the atherosclerotic lesions. Since the two subsets of T cells counteract each other, this switch may have important consequences for the inflammatory/immune process in atherosclerosis. PMID:9541503

Characteristic cytokine generation patterns in cancer cells and infiltrating lymphocytes in oral squamous cell carcinomas and the influence of chemoradiation combined with immunotherapy on these patterns.

PubMed

Yamamoto, Tetsuya; Kimura, Tsuyoshi; Ueta, Eisaku; Tatemoto, Yukihiro; Osaki, Tokio

2003-01-01

Cytokines produced by tumor cells and tumor-infiltrating lymphocytes (TIL) appear to regulate tumor cell growth and the cytotoxic activity of TIL. The objectives of the present study were to investigate cytokine generation patterns in tumor cells and TIL and to examine the influence of cancer therapy on this cytokine production and the cytotoxic activity of TIL. We determined the levels of cytokines produced by tumor cells and TIL in vitro and measured the cytotoxic activity of TIL against Daudi cells in patients with oral squamous cell carcinoma (OSC) before and 1 week after the start of concomitant chemo-radio-immunotherapy. Before the therapy, OSC cells generated higher levels of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) than did oral keratinocytes isolated from the noninflamed gingivae of healthy individuals, but both kinds of cells generated similar levels of interleukin (IL)-1beta and IL-6. Compared with peripheral blood mononuclear cells (PBMC) of the patients, TIL produced higher levels of IL-1beta, IL-6, IL-10, TNF-alpha and TGF-beta, whereas their production of IL-12 and interferon-gamma (IFN-gamma) was only slightly higher than that in PBMC. After 1 week of therapy, the cytokine production by OSC cells had largely decreased, while the production of TNF-alpha, IFN-gamma, TGF-beta and IL-12 by TIL had increased greatly, although other cytokine levels were almost constant during the investigations. The cytotoxic activity of TIL was higher than that of PBMC before the therapy, and this activity was strongly increased by 1 week of therapy. These results suggest that the cytokine productivities of TIL and tumor cells differ from those of PBMC and normal keratinocytes, respectively, and that chemo-radio-immunotherapy modulates in situ cytokine generation, which is advantageous for inhibition of tumor cell growth and activation of TIL. Copyright 2003 S. Karger AG, Basel

Design and interpretation of cell trajectory assays

PubMed Central

Bowden, Lucie G.; Simpson, Matthew J.; Baker, Ruth E.

2013-01-01

Cell trajectory data are often reported in the experimental cell biology literature to distinguish between different types of cell migration. Unfortunately, there is no accepted protocol for designing or interpreting such experiments and this makes it difficult to quantitatively compare different published datasets and to understand how changes in experimental design influence our ability to interpret different experiments. Here, we use an individual-based mathematical model to simulate the key features of a cell trajectory experiment. This shows that our ability to correctly interpret trajectory data is extremely sensitive to the geometry and timing of the experiment, the degree of motility bias and the number of experimental replicates. We show that cell trajectory experiments produce data that are most reliable when the experiment is performed in a quasi-one-dimensional geometry with a large number of identically prepared experiments conducted over a relatively short time-interval rather than a few trajectories recorded over particularly long time-intervals. PMID:23985736

A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages.

PubMed

Pearson, Bret J; Sánchez Alvarado, Alejandro

2010-01-01

The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. Here, we focus on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. Our results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal.

IL-17+ γδ T cells as kick-starters of inflammation.

PubMed

Papotto, Pedro H; Ribot, Julie C; Silva-Santos, Bruno

2017-05-18

Shortly after the discovery of interleukin 17 (IL-17)-producing CD4 + helper T cells (T H 17 cells), it was found that γδ T cells can also secrete large amounts of this pro-inflammatory cytokine. A decade later, it is now known that IL-17 + γδ T cells (γδ17 T cells) are often the main providers of IL-17A in various models of inflammatory diseases, while they also contribute to protective immune responses to infectious organisms. Due to an intricate thymic program of differentiation, γδ17 T cells are able to respond faster than T H 17 cells do and thus predominate in the early stages of inflammatory responses. Here we review the current knowledge of the development, activation and pathophysiological functions of γδ17 T cells, aiming to increase the awareness in the community of the therapeutic potential of this 'other side' of IL-17-mediated immune responses.

In vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A

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

Ramshaw, I.A.; Woodsworth, M.; Eidinger, D.

1979-01-01

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to BrMRBC. Mitogenic concentrations of Con A prevented themore » development of both the PFC and T/sub DTH/ responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and T/sub DTH/ response against BrMRBC.« less