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Sample records for rapidly dividing cells

  1. Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum

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    Vijay Swahari

    2016-01-01

    Full Text Available Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

  2. Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells

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    Teddy Léguillier

    2012-05-01

    Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

  3. Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion.

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    Gomes-Pereira, Mário; Hilley, James D; Morales, Fernando; Adam, Berit; James, Helen E; Monckton, Darren G

    2014-06-01

    Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenic mouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with a major role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. DNA repair mechanisms in dividing and non-dividing cells.

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    Iyama, Teruaki; Wilson, David M

    2013-08-01

    DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease. Published by Elsevier B.V.

  5. Flagellation of Pseudomonas aeruginosa in newly divided cells

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    Zhao, Kun; Lee, Calvin; Anda, Jaime; Wong, Gerard

    2015-03-01

    For monotrichous bacteria, Pseudomonas aeruginosa, after cell division, one daughter cell inherits the old flagellum from its mother cell, and the other grows a new flagellum during or after cell division. It had been shown that the new flagellum grows at the distal pole of the dividing cell when the two daughter cells haven't completely separated. However, for those daughter cells who grow new flagella after division, it still remains unknown at which pole the new flagellum will grow. Here, by combining our newly developed bacteria family tree tracking techniques with genetic manipulation method, we showed that for the daughter cell who did not inherit the old flagellum, a new flagellum has about 90% chances to grow at the newly formed pole. We proposed a model for flagellation of P. aeruginosa.

  6. Actin in dividing cells: contractile ring filaments bind heavy meromyosin.

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    Schroeder, T E

    1973-06-01

    Many microfilaments and microtubules are well preserved after glycerol-extraction of HeLa cells at room temperature (22 degrees ). Incubation in heavy meromyosin from rabbit skeletal muscle results in conspicuous and characteristic "decoration" of microfilaments of the contractile ring. Decoration is completely prevented by 10 mM ATP or 2 mM pyrophosphate, and fails to occur if heavy meromyosin is either omitted or replaced by egg albumin, a nonspecific protein. Decorated microfilaments have a substructure consisting of polarized, repeating arrowheads 27-35 nm apart. The specificity of these results strongly suggests that microfilaments of the contractile ring in HeLa cells are closely related to muscle actin. Very thin undecorated strands among the microfilaments of the contractile ring possibly represent a myosin component. These findings are discussed in terms of: the actomyosin-like properties of the contractile ring as a mechanochemical organelle that causes cell cleavage; the probable universal occurrence of actin-like protein in all dividing animal cells; and the contractile ring's combined sensitivity to cytochalasin B and its affinity for heavy meromyosin, a combination unique among microfilamentous organelles.

  7. Robustness of synthetic oscillators in growing and dividing cells

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    Paijmans, Joris; Lubensky, David K.; Rein ten Wolde, Pieter

    2017-05-01

    Synthetic biology sets out to implement new functions in cells, and to develop a deeper understanding of biological design principles. Elowitz and Leibler [Nature (London) 403, 335 (2000), 10.1038/35002125] showed that by rational design of the reaction network, and using existing biological components, they could create a network that exhibits periodic gene expression, dubbed the repressilator. More recently, Stricker et al. [Nature (London) 456, 516 (2008), 10.1038/nature07389] presented another synthetic oscillator, called the dual-feedback oscillator, which is more stable. Detailed studies have been carried out to determine how the stability of these oscillators is affected by the intrinsic noise of the interactions between the components and the stochastic expression of their genes. However, as all biological oscillators reside in growing and dividing cells, an important question is how these oscillators are perturbed by the cell cycle. In previous work we showed that the periodic doubling of the gene copy numbers due to DNA replication can couple not only natural, circadian oscillators to the cell cycle [Paijmans et al., Proc. Natl. Acad. Sci. (USA) 113, 4063 (2016), 10.1073/pnas.1507291113], but also these synthetic oscillators. Here we expand this study. We find that the strength of the locking between oscillators depends not only on the positions of the genes on the chromosome, but also on the noise in the timing of gene replication: noise tends to weaken the coupling. Yet, even in the limit of high levels of noise in the replication times of the genes, both synthetic oscillators show clear signatures of locking to the cell cycle. This work enhances our understanding of the design of robust biological oscillators inside growing and diving cells.

  8. Isolating dividing neural and brain tumour cells for gene expression profiling.

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    Endaya, Berwini; Cavanagh, Brenton; Alowaidi, Faisal; Walker, Tom; de Pennington, Nicholas; Ng, Jin-Ming A; Lam, Paula Y P; Mackay-Sim, Alan; Neuzil, Jiri; Meedeniya, Adrian C B

    2016-01-15

    The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Cortical microtubule labeling: Insight of AFH14 in non-dividing cells

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    Cai, Chao; Li, Yanhua; Shen, Yuan; Ren, Haiyun

    2010-01-01

    We recently reported that AFH14 participated in microtubule and actin filament interaction in cell division, and the AFH14 (FH1FH2) was important to the directly binding activity of microtubules and microfilaments. To preliminarily understand the function and localization of AFH14 in non-dividing cells, we overexpressed FH1FH2-RFP in onion epidermal cells, and found a fluorescence labeled filamentous network. The result of double labeling with different cytoskeleton reporter proteins indicate...

  10. Electrochemical treatment of reverse osmosis concentrate on boron-doped electrodes in undivided and divided cell configurations

    Energy Technology Data Exchange (ETDEWEB)

    Bagastyo, Arseto Y. [Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072 (Australia); Department of Environmental Engineering, Institut Teknologi Sepuluh Nopember, Surabaya 60111 (Indonesia); Batstone, Damien J. [Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072 (Australia); Kristiana, Ina [Curtin Water Quality Research Centre, Resources and Chemistry Precinct, Department of Chemistry, Curtin University, Bentley, Perth, WA 6102 (Australia); Escher, Beate I. [National Research Centre for Environmental Toxicology (Entox), The University of Queensland, Brisbane, QLD 4108 (Australia); Joll, Cynthia [Curtin Water Quality Research Centre, Resources and Chemistry Precinct, Department of Chemistry, Curtin University, Bentley, Perth, WA 6102 (Australia); Radjenovic, Jelena, E-mail: j.radjenovic@uq.edu.au [Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072 (Australia)

    2014-08-30

    Highlights: • 100% of COD and ∼70% of DOC was removed in both cell configurations. • ∼21.7 mg L{sup −1} of AOCl and ∼2.3 mg L{sup −1} of AOBr was formed regardless of the membrane use. • The TEQ was far lower than expected given the high AOCl concentrations. • The undivided cell consumed lower energy compared to the divided cell. - Abstract: An undivided electrolytic cell may offer lower electrochlorination through reduction of chlorine/hypochlorite at the cathode. This study investigated the performance of electrooxidation of reverse osmosis concentrate using boron-doped diamond electrodes in membrane-divided and undivided cells. In both cell configurations, similar extents of chemical oxygen demand and dissolved organic carbon removal were obtained. Continuous formation of chlorinated organic compounds was observed regardless of the membrane presence. However, halogenation of the organic matter did not result in a corresponding increase in toxicity (Vibrio fischeri bioassay performed on extracted samples), with toxicity decreasing slightly until 10 Ah L{sup −1}, and generally remaining near the initial baseline-toxicity equivalent concentration (TEQ) of the raw concentrate (i.e., ∼2 mg L{sup −1}). The exception was a high range toxicity measure in the undivided cell (i.e., TEQ = 11 mg L{sup −1} at 2.4 Ah L{sup −1}), which rapidly decreased to 4 mg L{sup −1}. The discrepancy between the halogenated organic matter and toxicity patterns may be a consequence of volatile and/or polar halogenated by-products formed in oxidation by OH· electrogenerated at the anode. The undivided cell exhibited lower energy compared to the divided cell, 0.25 kWh gCOD{sup −1} and 0.34 kWh gCOD{sup −1}, respectively, yet it did not demonstrate any improvement regarding by-products formation.

  11. Histone deacetylase inhibition activates transgene expression from integration-defective lentiviral vectors in dividing and non-dividing cells.

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    Pelascini, Laetitia P L; Janssen, Josephine M; Gonçalves, Manuel A F V

    2013-01-01

    Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone- and cell type-dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles.

  12. Electrochemical treatment of reverse osmosis concentrate on boron-doped electrodes in undivided and divided cell configurations.

    Science.gov (United States)

    Bagastyo, Arseto Y; Batstone, Damien J; Kristiana, Ina; Escher, Beate I; Joll, Cynthia; Radjenovic, Jelena

    2014-08-30

    An undivided electrolytic cell may offer lower electrochlorination through reduction of chlorine/hypochlorite at the cathode. This study investigated the performance of electrooxidation of reverse osmosis concentrate using boron-doped diamond electrodes in membrane-divided and undivided cells. In both cell configurations, similar extents of chemical oxygen demand and dissolved organic carbon removal were obtained. Continuous formation of chlorinated organic compounds was observed regardless of the membrane presence. However, halogenation of the organic matter did not result in a corresponding increase in toxicity (Vibrio fischeri bioassay performed on extracted samples), with toxicity decreasing slightly until 10AhL(-1), and generally remaining near the initial baseline-toxicity equivalent concentration (TEQ) of the raw concentrate (i.e., ∼2mgL(-1)). The exception was a high range toxicity measure in the undivided cell (i.e., TEQ=11mgL(-1) at 2.4AhL(-1)), which rapidly decreased to 4mgL(-1). The discrepancy between the halogenated organic matter and toxicity patterns may be a consequence of volatile and/or polar halogenated by-products formed in oxidation by OH electrogenerated at the anode. The undivided cell exhibited lower energy compared to the divided cell, 0.25kWhgCOD(-1) and 0.34kWhgCOD(-1), respectively, yet it did not demonstrate any improvement regarding by-products formation. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Integrating retroviral cassette extends gene delivery of HSV-1 expression vectors to dividing cells.

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    de Felipe, P; Izquierdo, M; Wandosell, F; Lim, F

    2001-08-01

    Retroviral vectors have long been used in a wide variety of gene transfer applications but have certain drawbacks, such as small cargo size, limited tropism, and low titers. HSV expression vectors overcome these disadvantages, but, because they persist in target cells as nonreplicative episomes, they are not retained in all the progeny of dividing cells. Chimeric HSV/AAV products that can mediate transgene integration in human mitotic cells have been constructed, but, to date, genetic modification of dividing cells in animal models using HSV products has not been possible. Here, we report the construction of hybrid HSV/retroviral vectors that exhibit up to 50-fold higher transgene integration efficiency compared to vectors containing only HSV-1 components. Efficient integration of a retroviral transgene cassette encoding pac in human cells required expression of the Moloney murine leukemia virus gag-pol genes, but in murine cells, could also be mediated by endogenous activities, albeit at a lower level. Gene delivery was equally efficient in BHK21, a cell line resistant to retroviral infection, and transgene retention and expression were observed to be stable for least one month in Hs683 human glioma cells. These vectors have wide applications for the genetic modification of many cell types.

  14. A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells.

    Directory of Open Access Journals (Sweden)

    Gilles Charvin

    Full Text Available BACKGROUND: Imaging single cells with fluorescent markers over multiple cell cycles is a powerful tool for unraveling the mechanism and dynamics of the cell cycle. Over the past ten years, microfluidic techniques in cell biology have emerged that allow for good control of growth environment. Yet the control and quantification of transient gene expression in unperturbed dividing cells has received less attention. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a microfluidic flow cell to grow Saccharomyces Cerevisiae for more than 8 generations (approximately 12 hrs starting with single cells, with controlled flow of the growth medium. This setup provides two important features: first, cells are tightly confined and grow in a remarkably planar array. The pedigree can thus be determined and single-cell fluorescence measured with 3 minutes resolution for all cells, as a founder cell grows to a micro-colony of more than 200 cells. Second, we can trigger and calibrate rapid and transient gene expression using reversible administration of inducers that control the GAL1 or MET3 promoters. We then show that periodic 10-20 minutes gene induction pulses can drive many cell division cycles with complete coherence across the cell cluster, with either a G1/S trigger (cln1 cln2 cln3 MET3-CLN2 or a mitotic trigger (cdc20 GALL-CDC20. CONCLUSIONS/SIGNIFICANCE: In addition to evident cell cycle applications, this device can be used to directly measure the amount and duration of any fluorescently scorable signal-transduction or gene-induction response over a long time period. The system allows direct correlation of cell history (e.g., hysteresis or epigenetics or cell cycle position with the measured response.

  15. Particle-in-cell simulations of electron beam control using an inductive current divider

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    Swanekamp, S. B.; Angus, J. R.; Cooperstein, G.; Ottinger, P. F.; Richardson, A. S.; Schumer, J. W.; Weber, B. V.

    2015-11-01

    Kinetic, time-dependent, electromagnetic, particle-in-cell simulations of the inductive current divider are presented. The inductive current divider is a passive method for controlling the trajectory of an intense, hollow electron beam using a vacuum structure that inductively splits the beam's return current. The current divider concept was proposed and studied theoretically in a previous publication [Swanekamp et al., Phys. Plasmas 22, 023107 (2015)]. A central post carries a portion of the return current (I1), while the outer conductor carries the remainder (I2) with the injected beam current given by Ib = I1 + I2. The simulations are in agreement with the theory which predicts that the total force on the beam trajectory is proportional to (I2-I1) and the force on the beam envelope is proportional to Ib. Independent control over both the current density and the beam angle at the target is possible by choosing the appropriate current-divider geometry. The root-mean-square (RMS) beam emittance (ɛRMS) varies as the beam propagates through the current divider to the target. For applications where control of the beam trajectory is desired and the current density at the target is similar to the current density at the entrance foil, there is a modest 20% increase in ɛRMS at the target. For other applications where the beam is pinched to a current density ˜5 times larger at the target, ɛRMS is 2-3 times larger at the target.

  16. Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos

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    Herbomel, Gaëtan; Hatte, Guillaume; Roul, Julien; Padilla-Parra, Sergi; Tassan, Jean-Pierre; Tramier, Marc

    2017-01-01

    Epithelia represent a unique situation where polarized cells must maintain sufficiently strong cell-cell contacts to guarantee the epithelial integrity indispensable for barrier functions. Nevertheless, epithelia must also keep sufficient plasticity which is crucial during development and morphogenesis. Adherens junctions and mechanical forces produced by the actomyosin cytoskeleton are major players for epithelial integrity maintenance and plasticity regulations. To understand how the epithelium is able to meet such a challenge, it is indispensable to determine how cellular junctions and mechanical forces acting at adherens junctions are regulated. Here, we investigate the tensile forces acting on adherens junctions via cadherin during cell division in the Xenopus embryos epithelium. Using the recently developed E-cadherin FRET tension sensor and a fastFLIM prototype microscope, we were able to measure mechanical forces applied on cadherin at cell-cell junctions. We have shown that the Xenopus epithelium is under tension, approximately 3 pN which remains stable, indicating that tensile forces acting on cadherin at the adherens junction are at equilibrium. Unexpectedly, mechanical tension across cadherin was similar between dividing and non-dividing epithelial cells. PMID:28327558

  17. The transforming parasite Theileria co-opts host cell mitotic and central spindles to persist in continuously dividing cells.

    Directory of Open Access Journals (Sweden)

    Conrad von Schubert

    2010-09-01

    Full Text Available The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.

  18. Biosynthesis of copper nanoparticles and its effect on actively dividing cells of mitosis in Allium cepa.

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    Nagaonkar, Dipali; Shende, Sudhir; Rai, Mahendra

    2015-01-01

    Nanobiotechnological application of copper nanoparticles has paved the way for advancement in agriculture owing to its bactericidal and fungicidal activities. Recently, researchers have focussed on bioinspired synthesis of copper nanoparticles as a viable alternative to existing physicochemical techniques. For the commercialization of nanocopper, the toxicity evaluation is a major issue. In this context, Citrus medica (L.) fruit extract-mediated copper nanoparticles were synthesized and its different concentrations (10, 20, 40, 60, 80, and 100 µg mL(-1) ) were evaluated for its effect on actively dividing cells of Allium cepa. The study clearly revealed that copper nanoparticles increased mitotic index up to the concentration of 20 µg mL(-1) . In addition, a gradual decline in mitotic index and increase in abnormality index was observed as the concentration of copper nanoparticles and treatment duration were increased. Aberrations in chromosomal behavior such as sticky and disturbed chromosomes in metaphase and anaphase, c-metaphase, bridges, laggard, disturbed telophase, and vacuolated nucleus were also observed. © 2015 American Institute of Chemical Engineers.

  19. SILICATE UPTAKE AND SILICA SHELL FORMATION BY SYNCHRONOUSLY DIVIDING CELLS OF THE DIATOM NAVICULA PELLICULOSA (BRÉB.) HILSE(1,2,3).

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    Busby, W F; Lewin, J

    1967-09-01

    Cells of the freshwater diatom Navicula pelliculosa were induced to divide synchronously by the addition of excess silicate (20 mg Si/liter) to a culture which had been silicate-starved for 14 hr. After a 3- to 4-hr lag period, characterized by rapid silicate uptake (6.5 mg Si/liter/4 hr) and silica shell formation, the cell number then doubled in the succeeding 2-3 hr. This system promises to have significance in the study of silicon metabolism, transport phenomena, and silica deposition.

  20. The molecularly crowded cytoplasm of bacterialcCells : Dividing cells contrasted with viable but non-culturable (VBNC) bacterial cells

    NARCIS (Netherlands)

    Trevors, J. T.; van Elsas, J. D.; Bej, A. K.

    2013-01-01

    In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but non-culturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell

  1. Activated leukocyte cell adhesion molecule (ALCAM/CD166): signaling at the divide of melanoma cell clustering and cell migration?

    NARCIS (Netherlands)

    Swart, G.W.M.; Lunter, P.C.; Kilsdonk, J.W.J. van; Kempen, L.C. van

    2005-01-01

    Orchestrated modulation of cell adhesion is essential for development and homeostasis in multicellular organisms. It optimizes embedding of the cell in its dynamic environment and facilitates appropriate cell responses and intercellular communication. Chronic disturbance of this delicate equilibrium

  2. Metabolic Profiling of Dividing Cells in Live Rodent Brain by Proton Magnetic Resonance Spectroscopy (1HMRS) and LCModel Analysis

    DEFF Research Database (Denmark)

    Park, June-Hee; Lee, Hedok; Makaryus, Rany

    2014-01-01

    RATIONALE: Dividing cells can be detected in the live brain by positron emission tomography or optical imaging. Here we apply proton magnetic resonance spectroscopy (1HMRS) and a widely used spectral fitting algorithm to characterize the effect of increased neurogenesis after electroconvulsive sh...

  3. Dividing Discipline

    DEFF Research Database (Denmark)

    Kristensen, Peter Marcus

    2012-01-01

    the periphery of the network—security studies and international political economy in particular—but communication is also divided along the lines of geography and policy/theory. The article concludes that divisions notwithstanding, IR communication remains centered around American, general, and theoretical IR...... than 20,000 articles published in 59 IR journals to construct a network among IR journals and finds a discipline with a center consisting of pedigreed IR journals, albeit closely related to political science. Divisions are identifiable as specialty areas that form clusters of specialized journals along...

  4. The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages

    Science.gov (United States)

    Guglielmi, Luca; Patzel, Eva; Sel, Saadettin; Auffarth, Gerd U.; Carl, Matthias; Poggi, Lucia

    2017-01-01

    Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP) transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC) containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers. PMID:28107513

  5. Regulation of the efflux of putrescine and cadaverine from rapidly growing cultured RAW 264 cells by extracellular putrescine.

    OpenAIRE

    Tjandrawinata, R R; Byus, C V

    1995-01-01

    Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acety...

  6. Why Cells Grow and Divide? General Growth Mechanism and How it Defines Cells’ Growth, Reproduction and Metabolic Properties

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    Shestopaloff, Yuri K.

    2015-02-01

    We consider a general growth mechanism, which acts at cellular level and above (organs, systems and whole organisms). Using its mathematical representation, the growth equation, we study the growth and division mechanisms of amoeba and fission yeast Schizosaccharomyces pombe. We show how this mechanism, together with biomolecular machinery, governs growth and reproduction of cells, and these organisms in particular. This mechanism provides revealing answers to fundamental questions of biology, like why cells grow and divide, why and when cells’ growth stops. It also sheds light on questions like why and how life originated and developed. Solving the growth equation, we obtain analytical expression for the growth curve of fission yeast as a function of geometrical characteristics and nutrient influxes for RNA and protein synthesis, and compare the computed growth curves with 85 experiments. Statistical evaluation shows that these growth curves correspond to experimental data significantly better than all previous approximations. Also, using the general growth mechanism, we show how metabolic characteristics of cells, their size and evolutionary traits relate, considering fission yeast. In particular, we found that fission yeast S. pombe consumes about 16-18 times more nutrients for maintenance needs than for biomass synthesis.

  7. The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

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    Collings, David A; Harper, John D I; Vaughn, Kevin C

    2003-12-01

    We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

  8. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

    Directory of Open Access Journals (Sweden)

    Smith Charlotte M

    2013-01-01

    Full Text Available Abstract Background During metaphase clathrin stabilises the mitotic spindle kinetochore(K-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

  9. Removal of Polyvinyl Alcohol in Aqueous Solutions Using an Innovative Paired Photoelectrochemical Oxidative System in a Divided Electrochemical Cell

    Directory of Open Access Journals (Sweden)

    Kai-Yu Huang

    2015-01-01

    Full Text Available This study evaluates the performance of an innovative paired photoelectrochemical oxidative system fabricated in our laboratory to determine the removal efficiency of polyvinyl alcohol (PVA in aqueous solutions. An innovative paired photoelectrochemical oxidative system employed metal redox mediators with high redox potential for anodic oxidation (MEO process and UV assisted photoelectrochemical oxidation (PEO process for cathodic oxidation in a divided electrochemical cell. Several parameters were investigated to characterize the removal efficiency of PVA, such as the current density, initial Ce(III concentration, nitric acid concentration, oxygen flow rate, and UV irradiation intensity. The effects of these parameters on the specific energy consumption were also investigated. Additionally, the conversion yield of Ce(IV concentration and the electrogeneration of H2O2 were calculated in this study. The optimum current density, initial Ce(III concentration, nitric acid concentration, oxygen flow rate, and UV irradiation intensity were found to be 3 mA cm−2, 0.01 M, 0.3 M, 500 cm3 min−1, and 1.2 mW cm−2, respectively. The synergistic effect of combination process of MEO and PEO would be as a promising alternative for the removal efficiency of PVA.

  10. Rapid magnetic cell delivery for large tubular bioengineered constructs.

    Science.gov (United States)

    Gonzalez-Molina, J; Riegler, J; Southern, P; Ortega, D; Frangos, C C; Angelopoulos, Y; Husain, S; Lythgoe, M F; Pankhurst, Q A; Day, R M

    2012-11-07

    Delivery of cells into tubular tissue constructs with large diameters poses significant spatial and temporal challenges. This study describes preliminary findings for a novel process for rapid and uniform seeding of cells onto the luminal surface of large tubular constructs. Fibroblasts, tagged with superparamagnetic iron oxide nanoparticles (SPION), were directed onto the luminal surface of tubular constructs by a magnetic field generated by a k4-type Halbach cylinder device. The spatial distribution of attached cells, as measured by the mean number of cells, was compared with a conventional, dynamic, rotational cell-delivery technique. Cell loading onto the constructs was measured by microscopy and magnetic resonance imaging. The different seeding techniques employed had a significant effect on the spatial distribution of the cells (p same construct was significantly different for the dynamic rotation technique (p delivery techniques and is amenable to a variety of tubular organs where rapid loading and uniform distribution of cells for therapeutic applications are required.

  11. Why do bacteria divide?

    Directory of Open Access Journals (Sweden)

    Vic eNorris

    2015-04-01

    Full Text Available The problem of not only how but also why cells divide can be tackled using recent ideas. One idea from the origins of life – Life as independent of its constituents – is that a living entity like a cell is a particular pattern of connectivity between its constituents. This means that if the growing cell were just to get bigger the average connectivity between its constituents per unit mass – its cellular connectivity – would decrease and the cell would lose its identity. The solution is division which restores connectivity. The corollary is that the cell senses decreasing cellular connectivity and uses this information to trigger division. A second idea from phenotypic diversity – Life on the Scales of Equilibria – is that a bacterium must find strategies that allow it to both survive and grow. This means that it has learnt to reconcile the opposing constraints that these strategies impose. The solution is that the cell cycle generates daughter cells with different phenotypes based on sufficiently complex equilibrium and non-equilibrium cellular compounds and structures appropriate for survival and growth, respectively, alias `hyperstructures'. The corollary is that the cell senses both the quantity of equilibrium material and the intensity of use of non-equilibrium material and then uses this information to trigger the cell cycle. A third idea from artificial intelligence – Competitive Coherence – is that a cell selects the active subset of elements that actively determine its phenotype from a much larger set of available elements. This means that the selection of an active subset of a specific size and composition must be done so as to generate both a coherent cell state, in which the cell’s contents work together harmoniously, and a coherent sequence of cell states, each coherent with respect to itself and to an unpredictable environment. The solution is the use of a range of mechanisms ranging from hyperstructure dynamics

  12. Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

    Science.gov (United States)

    Hoggatt, Jonathan; Singh, Pratibha; Tate, Tiffany A; Chou, Bin-Kuan; Datari, Shruti R; Fukuda, Seiji; Liu, Liqiong; Kharchenko, Peter V; Schajnovitz, Amir; Baryawno, Ninib; Mercier, Francois E; Boyer, Joseph; Gardner, Jason; Morrow, Dwight M; Scadden, David T; Pelus, Louis M

    2018-01-11

    Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROβ, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A rapid mitochondrial toxicity assay utilizing rapidly changing cell energy metabolism.

    Science.gov (United States)

    Sanuki, Yosuke; Araki, Tetsuro; Nakazono, Osamu; Tsurui, Kazuyuki

    2017-01-01

    Drug-induced liver injury is a major cause of safety-related drug-marketing withdrawals. Several drugs have been reported to disrupt mitochondrial function, resulting in hepatotoxicity. The development of a simple and effective in vitro assay to identify the potential for mitochondrial toxicity is thus desired to minimize the risk of causing hepatotoxicity and subsequent drug withdrawal. An in vitro test method called the "glucose-galactose" assay is often used in drug development but requires prior-culture of cells over several passages for mitochondrial adaptation, thereby restricting use of the assay. Here, we report a rapid version of this method with the same predictability as the original method. We found that replacing the glucose in the medium with galactose resulted in HepG2 cells immediately shifting their energy metabolism from glycolysis to oxidative phosphorylation due to drastic energy starvation; in addition, the intracellular concentration of ATP was reduced by mitotoxicants when glucose in the medium was replaced with galactose. Using our proposed rapid method, mitochondrial dysfunction in HepG2 cells can be evaluated by drug exposure for one hour without a pre-culture step. This rapid assay for mitochondrial toxicity may be more suitable for high-throughput screening than the original method at an early stage of drug development.

  14. Rapid induction of senescence in human cervical carcinoma cells

    Science.gov (United States)

    Goodwin, Edward C.; Yang, Eva; Lee, Chan-Jae; Lee, Han-Woong; Dimaio, Daniel; Hwang, Eun-Seong

    2000-09-01

    Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

  15. Rapid neurogenesis through transcriptional activation in human stem cells.

    Science.gov (United States)

    Busskamp, Volker; Lewis, Nathan E; Guye, Patrick; Ng, Alex H M; Shipman, Seth L; Byrne, Susan M; Sanjana, Neville E; Murn, Jernej; Li, Yinqing; Li, Shangzhong; Stadler, Michael; Weiss, Ron; Church, George M

    2014-11-17

    Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  16. High incidence of non-random template strand segregation and asymmetric fate determination in dividing stem cells and their progeny.

    Directory of Open Access Journals (Sweden)

    Michael J Conboy

    2007-05-01

    Full Text Available Decades ago, the "immortal strand hypothesis" was proposed as a means by which stem cells might limit acquiring mutations that could give rise to cancer, while continuing to proliferate for the life of an organism. Originally based on observations in embryonic cells, and later studied in terms of stem cell self-renewal, this hypothesis has remained largely unaccepted because of few additional reports, the rarity of the cells displaying template strand segregation, and alternative interpretations of experiments involving single labels or different types of labels to follow template strands. Using sequential pulses of halogenated thymidine analogs (bromodeoxyuridine [BrdU], chlorodeoxyuridine [CldU], and iododeoxyuridine [IdU], and analyzing stem cell progeny during induced regeneration in vivo, we observed extraordinarily high frequencies of segregation of older and younger template strands during a period of proliferative expansion of muscle stem cells. Furthermore, template strand co-segregation was strongly associated with asymmetric cell divisions yielding daughters with divergent fates. Daughter cells inheriting the older templates retained the more immature phenotype, whereas daughters inheriting the newer templates acquired a more differentiated phenotype. These data provide compelling evidence of template strand co-segregation based on template age and associated with cell fate determination, suggest that template strand age is monitored during stem cell lineage progression, and raise important caveats for the interpretation of label-retaining cells.

  17. Continental Divide Trail

    Data.gov (United States)

    Earth Data Analysis Center, University of New Mexico — This shapefile was created to show the proximity of the Continental Divide to the Continental Divide National Scenic Trail in New Mexico. This work was done as part...

  18. Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells.

    Directory of Open Access Journals (Sweden)

    James C Walsh

    Full Text Available Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo.

  19. Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells.

    Science.gov (United States)

    Brunati, Cristina; Thomsen, Thomas T; Gaspari, Eleonora; Maffioli, Sonia; Sosio, Margherita; Jabes, Daniela; Løbner-Olesen, Anders; Donadio, Stefano

    2017-10-30

    To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria. We used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time-kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles. The results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages. Overall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance.

  20. Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum.

    Science.gov (United States)

    Winnicki, Konrad; Żabka, Aneta; Bernasińska, Joanna; Matczak, Karolina; Maszewski, Janusz

    2015-06-01

    In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50% of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species.

  1. Rapid Turnover of Effector–Memory CD4+ T Cells in Healthy Humans

    Science.gov (United States)

    Macallan, Derek C.; Wallace, Diana; Zhang, Yan; de Lara, Catherine; Worth, Andrew T.; Ghattas, Hala; Griffin, George E.; Beverley, Peter C.L.; Tough, David F.

    2004-01-01

    Memory T cells can be divided into central–memory (TCM) and effector–memory (TEM) cells, which differ in their functional properties. Although both subpopulations can persist long term, it is not known whether they are maintained by similar mechanisms. We used in vivo labeling with deuterated glucose to measure the turnover of CD4+ T cells in healthy humans. The CD45R0+CCR7− TEM subpopulation was shown to have a rapid proliferation rate of 4.7% per day compared with 1.5% per day for CD45R0+CCR7+ TCM cells; these values are equivalent to average intermitotic (doubling) times of 15 and 48 d, respectively. In contrast, the CD45RA+CCR7+ naive CD4+ T cell population was found to be much longer lived, being labeled at a rate of only 0.2% per day (corresponding to an intermitotic time of approximately 1 yr). These data indicate that human CD4+ TEM cells constitute a short-lived cell population that requires continuous replenishment in vivo. PMID:15249595

  2. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  3. Rapid flow-induced responses in endothelial cells

    Science.gov (United States)

    Stamatas, G. N.; McIntire, L. V.

    2001-01-01

    Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

  4. International Divider Walls

    NARCIS (Netherlands)

    Kruis, A.; Sneller, A.C.W.(L.)

    2013-01-01

    The subject of this teaching case is the Enterprise Resource Planning (ERP) system implementation at International Divider Walls, the world market leader in design, production, and sales of divider walls. The implementation in one of the divisions of this multinational company had been successful,

  5. Rapid thermal sintering of the metallizations of silicon solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Laugier, A.; El Omari, H.; Boyeaux, J.P. [Institut National des Sciences Appliquees de Lyon, Villeurbanne (France). Lab. de Physique de la Matiere; Hartiti, B.; Muller, J.C. [CNRS, Strasbourg (France). Lab. de Physique et Applications des Semiconducteurs; Nam, L.Q.; Sarti, D. [Photowatt International S.A., Bourgoin Jallieu (France)

    1994-12-31

    Rapid Thermal Processing (RTP) using radiation from tungsten halogen lamps as a heat source is a very promising candidate to replace conventional furnace annealing as it offers many advantages such as a reduced overall thermal budget and a lower gas consumption. In this paper the authors show that with moderate temperature, RTP can be used to obtain screen printed contacts with low contacts resistivity and without degrading the transport properties of the solar cell base region. They investigate on Polix multicrystalline solar cells the possibility to replace the conventional sintering by a RTP annealing of the Ag front grid and of the back Al/Ag contact in a single step performed after the antireflection coating deposition.

  6. Designing Multicomponent Nanosystems for Rapid Detection of Circulating Tumor Cells.

    Science.gov (United States)

    Banerjee, Shashwat S; Khobragade, Vrushali; Khandare, Jayant

    2017-01-01

    Detection of circulating tumor cells (CTCs) in the blood circulation holds immense promise as it predicts the overall probability of patient survival. Therefore, CTC-based technologies are gaining prominence as a "liquid biopsy" for cancer diagnostics and prognostics. Here, we describe the design and synthesis of two distinct multicomponent magnetic nanosystems for rapid capture and detection of CTCs. The multifunctional Magneto-Dendrimeric Nano System (MDNS) composed of an anchoring dendrimer that is conjugated to multiple agents such as near infrared (NIR) fluorescent cyanine 5 NHS (Cy5), glutathione (GSH), transferrin (Tf), and iron oxide (Fe3O4) magnetic nanoparticle (MNP) for simultaneous tumor cell-specific affinity, multimodal high resolution confocal imaging, and cell isolation. The second nanosystem is a self-propelled microrocket that is composed of carbon nanotube (CNT), chemically conjugated with targeting ligand such as transferrin on the outer surface and Fe3O4 nanoparticles in the inner surface. The multicomponent nanosystems described here are highly efficient in targeting and isolating cancer cells thus benefiting early diagnosis and therapy of cancer.

  7. Parenteral nutrition rapidly reduces hepatic mononuclear cell numbers and lipopolysaccharide receptor expression on Kupffer cells in mice.

    Science.gov (United States)

    Omata, Jiro; Fukatsu, Kazuhiko; Murakoshi, Satoshi; Noguchi, Midori; Moriya, Tomoyuki; Okamoto, Koichi; Saitoh, Daizoh; Yamamoto, Junji; Hase, Kazuo

    2010-01-01

    Parenteral nutrition (PN) reduces the number of hepatic mononuclear cell (MNCs) and impairs their function, resulting in poor survival after intraportal bacterial challenge in mice. Our recent animal study demonstrated resumption of enteral nutrition after PN to rapidly restore hepatic MNC numbers (in 12 hours) and lipopolysaccharide (LPS) receptor expression on Kupffer cells (in 48 hours). The present study examined the time courses of hepatic MNC number reductions and LPS receptor expression changes in mice receiving PN. Male mice (n = 49) from the Institute of Cancer Research were divided into chow (n = 8), PN0.5 (n = 8), PN1 (n = 8), PN2 (n = 9), PN3 (n = 9), and PN5 (n = 7) groups. The chow group was given chow with an intravenous saline infusion. The PN groups were fed parenterally for 0.5, 1, 2, 3, or 5 days following the chow-feeding courses. After 7 days of nutrition support, hepatic MNCs were isolated and counted. The expression of LPS receptors on Kupffer cells was analyzed by flow cytometry. Hepatic MNC numbers rapidly reached their lowest level in the PN0.5 and PN1 groups but were somewhat restored thereafter and remained stable after the third day, without significant differences between any 2 of the PN groups. CD14 and Toll-like receptor 4/MD-2 expressions both showed significant reductions in the PN1 group compared with the chow group and gradually decreased to their lowest levels in the PN5 group. PN administration rapidly reduces hepatic MNC numbers and LPS receptor expression on Kupffer cells.

  8. Plasmonic cell nanocoating: a new concept for rapid microbial screening.

    Science.gov (United States)

    Xu, Ke; Bui, Minh-Phuong N; Fang, Aiqin; Abbas, Abdennour

    2017-11-01

    Nanocoating of single microbial cells with gold nanostructures can confer optical, electrical, thermal, and mechanical properties to microorganisms, thus enabling new avenues for their control, study, application, and detection. Cell nanocoating is often performed using layer-by-layer (LbL) deposition. LbL is time-consuming and relies on nonspecific electrostatic interactions, which limit potential applications for microbial diagnostics. Here, we show that, by taking advantage of surface molecules densely present in the microbial outer layers, cell nanocoating with gold nanoparticles can be achieved within seconds using surface molecules, including disulfide- bond-containing (Dsbc) proteins and chitin. A simple activation of these markers and their subsequent interaction with gold nanoparticles allow specific microbial screening and quantification of bacteria and fungi within 5 and 30 min, respectively. The use of plasmonics and fluorescence as transduction methods offers a limit of detection below 35 cfu mL-1 for E. coli bacteria and 1500 cfu mL-1 for M. circinelloides fungi using a hand-held fluorescent reader. Graphical abstract A new concept for rapid microbial screening by targeting disulfide - bond-containing (Dsbc) proteins and chitin with reducing agents and gold nanoparticles.

  9. Optimized Tetrazine Derivatives for Rapid Bioorthogonal Decaging in Living Cells.

    Science.gov (United States)

    Fan, Xinyuan; Ge, Yun; Lin, Feng; Yang, Yi; Zhang, Gong; Ngai, William Shu Ching; Lin, Zhi; Zheng, Siqi; Wang, Jie; Zhao, Jingyi; Li, Jie; Chen, Peng R

    2016-11-02

    The inverse-electron-demand Diels-Alder (iDA) reaction has recently been repurposed as a bioorthogonal decaging reaction by accelerating the elimination process after an initial cycloaddition between trans-cyclooctene (TCO) and tetrazine (TZ). Herein, we systematically surveyed 3,6-substituted TZ derivatives by using a fluorogenic TCO-coumarin reporter followed by LC-MS analysis, which revealed that the initial iDA cycloaddition step was greatly accelerated by electron-withdrawing groups (EWGs) while the subsequent elimination step was strongly suppressed by EWGs. In addition, smaller substituents facilitated the decaging process. These findings promoted us to design and test unsymmetric TZs bearing an EWG group and a small non-EWG group at the 3- and 6-position, respectively. These TZs showed remarkably enhanced decaging rates, enabling rapid iDA-mediated protein activation in living cells. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. The Open Access Divide

    Directory of Open Access Journals (Sweden)

    Jingfeng Xia

    2013-10-01

    Full Text Available This paper is an attempt to review various aspects of the open access divide regarding the difference between those academics who support free sharing of data and scholarly output and those academics who do not. It provides a structured description by adopting the Ws doctrines emphasizing such questions as who, what, when, where and why for information-gathering. Using measurable variables to define a common expression of the open access divide, this study collects aggregated data from existing open access as well as non-open access publications including journal articles and extensive reports. The definition of the open access divide is integrated into the discussion of scholarship on a larger scale.

  11. A rapid method of fruit cell isolation for cell size and shape measurements

    Directory of Open Access Journals (Sweden)

    Johnston Jason W

    2009-04-01

    Full Text Available Abstract Background Cell size is a structural component of fleshy fruit, contributing to important traits such as fruit size and texture. There are currently a number of methods for measuring cell size; most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes or chemicals to release single cells. Neither of these approaches is ideal for assaying large fruit numbers as both require a considerable time to prepare the tissue, with current methods of cell wall digestions taking 24 to 48 hours. Additionally, sectioning can lead to a measurement of a plane that does not represent the widest point of the cell. Results To develop a more rapid way of measuring fruit cell size we have developed a protocol that solubilises pectin in the middle lamella of the plant cell wall releasing single cells into a buffered solution. Gently boiling small fruit samples in a 0.05 M Na2CO3 solution, osmotically balanced with 0.3 M mannitol, produced good cell separation with little cellular damage in less than 30 minutes. The advantage of combining a chemical treatment with boiling is that the cells are rapidly killed. This stopped cell shape changes that could potentially occur during separation. With this method both the rounded and angular cells of the apple cultivars SciRos 'Pacific Rose' and SciFresh 'Jazz'™ were observed in the separated cells. Using this technique, an in-depth analysis was performed measuring cell size from 5 different apple cultivars. Cell size was measured using the public domain ImageJ software. For each cultivar a minimum of 1000 cells were measured and it was found that each cultivar displayed a different distribution of cell size. Cell size within cultivars was similar and there was no correlation between flesh firmness and cell size. This protocol was tested on tissue from other fleshy fruit including tomato, rock melon and kiwifruit. It was found that good cell separation was achieved with flesh

  12. Genome-wide screen identifies pathways that govern GAA/TTC repeat fragility and expansions in dividing and nondividing yeast cells.

    Science.gov (United States)

    Zhang, Yu; Shishkin, Alexander A; Nishida, Yuri; Marcinkowski-Desmond, Dana; Saini, Natalie; Volkov, Kirill V; Mirkin, Sergei M; Lobachev, Kirill S

    2012-10-26

    Triplex structure-forming GAA/TTC repeats pose a dual threat to the eukaryotic genome integrity. Their potential to expand can lead to gene inactivation, the cause of Friedreich's ataxia disease in humans. In model systems, long GAA/TTC tracts also act as chromosomal fragile sites that can trigger gross chromosomal rearrangements. The mechanisms that regulate the metabolism of GAA/TTC repeats are poorly understood. We have developed an experimental system in the yeast Saccharomyces cerevisiae that allows us to systematically identify genes crucial for maintaining the repeat stability. Two major groups of mutants defective in DNA replication or transcription initiation are found to be prone to fragility and large-scale expansions. We demonstrate that problems imposed by the repeats during DNA replication in actively dividing cells and during transcription initiation in nondividing cells can culminate in genome instability. We propose that similar mechanisms can mediate detrimental metabolism of GAA/TTC tracts in human cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. A dividing issue

    DEFF Research Database (Denmark)

    Gamborg, Christian; Jensen, Frank Søndergaard; Sandøe, Peter

    2016-01-01

    Why are organised shoots involving birds that are farm-reared and subsequently released a dividing issue in several countries? As a contribution to answering this question the paper reports a national survey of landowners (n = 1207), hunters (n = 1130) and the general public (n = 1001) in Denmark....... While there was broad agreement across all three groups that recreational hunting of naturally occurring “surplus” wildlife is acceptable, the release of farm-reared game birds for shooting was a dividing issue, both within the groups and between them. The majority of participants (51%) in the survey...... representing the general public were against the practice; a majority of hunters (61%) were in favour of it; and landowner approval rates lay between these two poles. Respondents with a “mutualist” or “distanced” wildlife value orientation according to the definitions by Teel et al. (2005) consistently...

  14. Facebook's gender divide

    OpenAIRE

    Garcia, David; Kassa, Yonas Mitike; Cuevas, Angel; Cebrian, Manuel; Moro, Esteban; Rahwan, Iyad; Cuevas, Ruben

    2017-01-01

    Online social media are information resources that can have a transformative power in society. While the Web was envisioned as an equalizing force that allows everyone to access information, the digital divide prevents large amounts of people from being present online. Online social media in particular are prone to gender inequality, an important issue given the link between social media use and employment. Understanding gender inequality in social media is a challenging task due to the neces...

  15. Melting the Divide

    Science.gov (United States)

    Gibson, S. M.

    2014-12-01

    Presenting Quaternary Environmental Change to students who fall into Widening Participation criteria at the University of Cambridge, gives a unique opportunity to present academic debate in an approachable and entertaining way. Literally by discussing the melting of our ice caps, melts the divide Cambridge has between its reputation and the reality for the brightest, underprivileged, students. There is a balance between presenting cutting edge research with the need to come across as accessible (and importantly valuable to "learning"). Climate change over the Quaternary lends itself well to this aim. By lecturing groups of potential students through the entire Quaternary in an hour, stopping to discuss how our ancestors interacted with past Interglacials and what are the mechanisms driving change (in generalized terms), you are able to introduce cutting edge research (such as the latest NEEM ice core) to the students. This shows the evolution and importance of higher education and academic research. The lecture leads well onto group discussions (termed "supervisions" in Cambridge), to explore their opinions on the concern for present Anthropogenic Climate Change in relation to Past Climate Change after being presented with images that our ancestors "made it". Here discussion thrives off students saying obvious things (or sarcastic comments!) which quickly can lead into a deep technical discussion on their terms. Such discussions give the students a zest for higher education, simply throwing Ruddiman's (2003) "The Anthroprocene Started Several Thousand Years Ago" at them, questions in a second their concept of Anthropogenic Climate Change. Supervisions lend themselves well to bright, articulate, students and by offering these experiences to students of Widening Participation criteria we quickly melt the divide between the reputation of Cambridge ( and higher education as a whole) and the day to day practice. Higher education is not for the privileged, but a free and

  16. Divide and Pacify

    DEFF Research Database (Denmark)

    Vanhuysse, Pieter

    unemployment benefits and many others onto early retirement and disability pensions. From a narrow economic viewpoint, these policies often appeared to be immensely costly or irresponsibly populist. Yet a more inclusive social-scientific perspective can shed new light on these seemingly irrational policies......, hundreds of thousands of people suddenly became disabled and retired or simply vanished from the labor market. … Pieter Vanhuysse, in his lucid and brilliantly argued slim book Divide and Pacify, shows how the second oddity goes a long way toward explaining the first one. By connecting two puzzles he...... about political economy as an art of analysis. Vanhuysse presents theory, methodology and empirical analysis with equal force, blends them into a powerful and very interesting narrative…. Highly recommended" – European Sociological Review "Pieter Vanhuysse has written a fascinating book on the phenomena...

  17. The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano.

    Science.gov (United States)

    Egger, Bernhard; Gschwentner, Robert; Hess, Michael W; Nimeth, Katharina T; Adamski, Zbigniew; Willems, Maxime; Rieger, Reinhard; Salvenmoser, Willi

    2009-07-15

    Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts) in S-phase. The tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells), was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells), while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point. Regeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before differentiation, in contrast to progenitor cells of

  18. Rapid white blood cell detection for peritonitis diagnosis

    Science.gov (United States)

    Wu, Tsung-Feng; Mei, Zhe; Chiu, Yu-Jui; Cho, Sung Hwan; Lo, Yu-Hwa

    2013-03-01

    A point-of-care and home-care lab-on-a-chip (LoC) system that integrates a microfluidic spiral device as a concentrator with an optical-coding device as a cell enumerator is demonstrated. The LoC system enumerates white blood cells from dialysis effluent of patients receiving peritoneal dialysis. The preliminary results show that the white blood cell counts from our system agree well with the results from commercial flow cytometers. The LoC system can potentially bring significant benefits to end stage renal disease (ESRD) patients that are on peritoneal dialysis (PD).

  19. T cells suppress memory-dependent rapid mucous cell metaplasia in mouse airways.

    Science.gov (United States)

    Chand, Hitendra S; Mebratu, Yohannes A; Montera, Marena; Tesfaigzi, Yohannes

    2016-10-20

    Airway epithelial cells (AECs) are crucial for mucosal and adaptive immunity but whether these cells respond in a memory-dependent manner is poorly studied. Previously, we have reported that LPS intratracheal instillation in rodents causes extensive neutrophilic inflammation and airway epithelial cell hyperplasia accompanied by mucous cell metaplasia (MCM). And the resolution process required a period of 40 d for the inflammation to subside and the lung epithelia to resemble the non-exposed condition. Therefore, the present study investigated the memory-dependent response of airway epithelial cells to a secondary LPS challenge after the initial inflammation was resolved. Airway epithelial and mucous cells were assessed in response to a secondary LPS challenge in F344/N rats, and in C57BL/6 wild-type (Foxn1(WT)) and T cell-deficient athymic (Foxn1(nu)) mice that were instilled with LPS or saline 40 d earlier. Epithelial expression of TLR4, EGFR, and phosphorylated-ERK1/2 (pERK) were also analyzed. LPS-pretreated F344/N rats responded with elevated numbers of AECs after saline challenge and with 3-4-fold increased MCM following the LPS challenge in LPS- compared with saline-pretreated rats. LPS-pretreated rats showed 5-fold higher number of AECs expressing TLR4 apically than saline-pretreated rats. Also, the expression of EGFR was increased in LPS-pretreated rats along with the number of AECs with active or nuclear pERK, and the levels were further increased upon LPS challenge. LPS-pretreated Foxn1(nu) compared with Foxn1(WT) mice showed increased MCM and elevated levels of TLR4, EGFR, and nuclear pERK at 40 d after LPS instillation. LPS challenge further augmented MCM rapidly in Foxn1(nu) compared with Foxn1(WT) mice. Together, these data suggest that AECs preserve an 'innate memory' that drives a rapid mucous phenotype via spatiotemporal regulation of TLR4 and EGFR. Further, T cells may suppress the sustained elevated expression of TLR4 and EGFR and thereby the

  20. CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability

    NARCIS (Netherlands)

    van der Windt, Gerritje J. W.; O'Sullivan, David; Everts, Bart; Huang, Stanley Ching-Cheng; Buck, Michael D.; Curtis, Jonathan D.; Chang, Chih-Hao; Smith, Amber M.; Ai, Teresa; Faubert, Brandon; Jones, Russell G.; Pearce, Edward J.; Pearce, Erika L.

    2013-01-01

    A characteristic of memory T (T-M) cells is their ability to mount faster and stronger responses to reinfection than naive T (T-N) cells do in response to an initial infection. However, the mechanisms that allow this rapid recall are not completely understood. We found that CD8 T-M cells have more

  1. Haemolysis following rapid experimental red blood cell transfusion--an evaluation of two infusion pumps

    DEFF Research Database (Denmark)

    Hansen, Tom Giedsing; Sprogøe-Jakobsen, U; Pedersen, C M

    1998-01-01

    The vast majority of infusion pumps used for rapid transfusion of large amounts of blood have never been properly examined regarding their influence on the quality of the red blood cells (RBCs) infused. In this study, we evaluated the effect of two different infusion pumps on the degree of RBC...... destruction following rapid experimental blood transfusion....

  2. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor

    Science.gov (United States)

    2012-01-01

    Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols. PMID:22475724

  3. IL-6 trans-Signaling-Dependent Rapid Development of Cytotoxic CD8+ T Cell Function

    Directory of Open Access Journals (Sweden)

    Jan P. Böttcher

    2014-09-01

    Full Text Available Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8+ T cells that use granzyme B (GzmB for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6 trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8+ T cells. Such LSEC-stimulated GzmB-expressing CD8+ T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8+ T cells that may be beneficial for vaccination strategies.

  4. Rapid cell-surface prion protein conversion revealed using a novel cell system

    Science.gov (United States)

    Goold, R.; Rabbanian, S.; Sutton, L.; Andre, R.; Arora, P.; Moonga, J.; Clarke, A.R.; Schiavo, G.; Jat, P.; Collinge, J.; Tabrizi, S.J.

    2011-01-01

    Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion. PMID:21505437

  5. Subsets of CD34+ cells and rapid hematopoietic recovery after peripheral-blood stem-cell transplantation

    NARCIS (Netherlands)

    Dercksen, M. W.; Rodenhuis, S.; Dirkson, M. K.; Schaasberg, W. P.; Baars, J. W.; van der Wall, E.; Slaper-Cortenbach, I. C.; Pinedo, H. M.; von dem Borne, A. E.; van der Schoot, C. E.

    1995-01-01

    To study whether there is a relationship between transplanted cell dose and rate of hematopoietic recovery after peripheral-blood stem-cell (PBSC) transplantation, and to obtain an indication whether specific subsets of CD34+ cell populations contribute to rapid recovery of neutrophils or platelets.

  6. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R

    2003-01-01

    to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...... neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system...

  7. Rapid prototyping methods for the manufacture of fuel cells

    Directory of Open Access Journals (Sweden)

    Dudek Piotr

    2016-01-01

    The potential for the application of this method for the manufacture of metallic bipolar plates (BPP for use in proton exchange membrane fuel cells (PEMFCs is presented and discussed. Special attention is paid to the fabrication of light elements for the construction of PEMFC stacks designed for mobile applications such as aviation technology and unmanned aerial vehicles (UAVs.

  8. Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose.

    Science.gov (United States)

    Macallan, Derek C; Asquith, Becca; Zhang, Yan; de Lara, Catherine; Ghattas, Hala; Defoiche, Julien; Beverley, Peter C L

    2009-01-01

    Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water ((2)H(2)O). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-(2)H(2)]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10-24 h; sampling, approximately 3 weeks; DNA extraction/derivatization, 2-3 d; and GC/MS analysis, approximately 2 d.

  9. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow

    Science.gov (United States)

    Colter, David C.; Class, Reiner; DiGirolamo, Carla M.; Prockop, Darwin J.

    2000-01-01

    Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm2) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 109-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation. PMID:10725391

  10. NF-κB is activated in CD4+ iNKT cells by sickle cell disease and mediates rapid induction of adenosine A2A receptors.

    Directory of Open Access Journals (Sweden)

    Gene Lin

    Full Text Available Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT cells. Sickle cell disease (SDC results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs. Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65 and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2 during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.

  11. Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imaging.

    Science.gov (United States)

    Boudreault, F; Grygorczyk, R

    2004-09-01

    Precise measurement of rapid volume changes of substrate-adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate-adherent cells based on phase-contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom-designed, side-viewing chamber and subjected to hypotonic swelling. Side-view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off-line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (DeltaVolume microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.

  12. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

    Science.gov (United States)

    Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

    2018-02-12

    Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

  13. Rapid and Efficient Generation of Regulatory T Cells to Commensal Antigens in the Periphery

    Directory of Open Access Journals (Sweden)

    Katherine Nutsch

    2016-09-01

    Full Text Available Commensal bacteria shape the colonic regulatory T (Treg cell population required for intestinal tolerance. However, little is known about this process. Here, we use the transfer of naive commensal-reactive transgenic T cells expressing colonic Treg T cell receptors (TCRs to study peripheral Treg (pTreg cell development in normal hosts. We found that T cells were activated primarily in the distal mesenteric lymph node. Treg cell induction was rapid, generating >40% Foxp3+ cells 1 week after transfer. Contrary to prior reports, Foxp3+ cells underwent the most cell divisions, demonstrating that pTreg cell generation can be the dominant outcome from naive T cell activation. Moreover, Notch2-dependent, but not Batf3-dependent, dendritic cells were involved in Treg cell selection. Finally, neither deletion of the conserved nucleotide sequence 1 (CNS1 region in Foxp3 nor blockade of TGF-β (transforming growth factor-β-receptor signaling completely abrogated Foxp3 induction. Thus, these data show that pTreg cell selection to commensal bacteria is rapid, is robust, and may be specified by TGF-β-independent signals.

  14. Fundamentals of rapid injection molding for microfluidic cell-based assays.

    Science.gov (United States)

    Lee, Ulri N; Su, Xiaojing; Guckenberger, David J; Dostie, Ashley M; Zhang, Tianzi; Berthier, Erwin; Theberge, Ashleigh B

    2018-01-30

    Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. 'Rapid' injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

  15. The digital divide: philosophical reflection

    Directory of Open Access Journals (Sweden)

    Dedyulina Marina Anatolevna

    2017-07-01

    Full Text Available The problem of digital divide itself is interesting for philosophical reflection as it lies at the crossroads of interests of social and political philosophy, philosophy of technology and epistemology, and these are just some of them. Due to the constant development of information technologies and the introduction of new technologies the digital divide is a dynamic problem. The main aim of this work is to analyse the conceptual and descriptive aspects of the problem of the digital divide, to get a more complete picture of the phenomenon. The digital divide is a complex problem that has social, political, cultural and ethical aspects.

  16. Near infrared photoimmunotherapy rapidly elicits specific host immunity against cancer cells (Conference Presentation)

    Science.gov (United States)

    Kobayashi, Hisataka

    2017-02-01

    Near infrared photoimmunotherapy (NIR-PIT) is a new molecularly-targeted cancer photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (mAb) targeting cell-surface molecules. When exposed to NIR light, the conjugate induces a highly-selective necrotic/immunogenic cell death (ICD) only in target-positive, mAb-IR700-bound cancer cells. This cell death occurs as early as 1 minute after exposure to NIR light. Meanwhile, immediately adjacent target-negative cells are unharmed. Dynamic 3D-microscopy of live tumor cells undergoing NIR-PIT showed rapid swelling in treated cells immediately after light exposure, followed by irreversible morphologic changes such as bleb formation, and rupture of vesicles within several minutes. Furthermore, biological markers of ICD including relocation of HSP70/90 and calreticulin, and release of ATP and High Mobility Group Box 1 (HMGB1), were clearly detected immediately after NIR-PIT. When NIR-PIT was performed in a mixture of cancer cells and immature dendritic cells, maturation of immature dendritic cells was strongly induced rapidly after NIR-PIT. Alternatively, NIR-PIT can also target negative regulatory immune cells such as Treg only in the tumor bed. Treg targeting NIR-PIT against CD25 can deplete >80% of Treg in tumor bed within 20 min that induces activation of tumor cell-specific CD8+-T and NK cells within 1.5 hour, and then these activated cells killed cancer cells in local tumor within 1 day and also in distant tumors of the same cell origin within 2 days. In summary, cancer cell-targeting and immuno-suppressor cell-targeting NIR-PITs effectively induce innate and acquired immunity specifically against cancer cells growing in patients, respectively.

  17. The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

    Directory of Open Access Journals (Sweden)

    Adamski Zbigniew

    2009-07-01

    Full Text Available Abstract Background Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts in S-phase. Results The tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells, was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells, while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point. Conclusion Regeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before

  18. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  19. Hyperkalemia caused by rapid red cell transfusion and the potassium absorption filter

    Directory of Open Access Journals (Sweden)

    Yasuhiko Imashuku

    2017-01-01

    Full Text Available We report a case of transient hyperkalemia during hysterectomy after cesarean section, due to preoperatively undiagnosed placenta accreta that caused unforeseen massive hemorrhage and required rapid red cell transfusion. Hyperkalemia-induced by rapid red cell transfusion is a well-known severe complication of transfusion; however, in patients with sudden massive hemorrhage, rapid red cell transfusion is necessary to save their life. In such cases, it is extremely important to monitor serum potassium levels. For an emergency situation, a system should be developed to ensure sufficient preparation for immediate transfusion and laboratory tests. Furthermore, sufficient stock of preparations to treat hyperkalemia, such as calcium preparations, diuretics, glucose, and insulin is required. Moreover, a transfusion filter that absorbs potassium has been developed and is now available for clinical use in Japan. The filter is easy to use and beneficial, and should be prepared when it is available.

  20. The oncogene c-Myc coordinates regulation of metabolic networks to enable rapid cell cycle entry.

    Science.gov (United States)

    Morrish, Fionnuala; Neretti, Nicola; Sedivy, John M; Hockenbery, David M

    2008-04-15

    The c-myc proto-oncogene is rapidly activated by serum and regulates genes involved in metabolism and cell cycle progression. This gene is thereby uniquely poised to coordinate both the metabolic and cell cycle regulatory events required for cell cycle entry. However, this function of Myc has not been evaluated. Using a rat fibroblast model of isogenic cell lines, myc(-/-), myc(+/-), myc(+/+) and myc(-/-) cells with an inducible c-myc transgene (mycER), we show that the Myc protein programs cells to utilize both oxidative phosphorylation and glycolysis to drive cell cycle progression. We demonstrate this coordinate regulation of metabolic networks is essential, as specific inhibitors of these pathways block Myc-induced proliferation. Metabolic events temporally correlated with cell cycle entry include increased oxygen consumption, mitochondrial function, pyruvate and lactate production, and ATP generation. Treatment of normal cells with inhibitors of oxidative phosphorylation recapitulates the myc(-/-) phenotype, resulting in impaired cell cycle entry and reduced metabolism. Combined with a kinetic expression profiling analysis of genes linked to mitochondrial function, our study indicates that Myc's ability to coordinately regulate the mitochondrial metabolic network transcriptome is required for rapid cell cycle entry. This function of Myc may underlie the pervasive presence of Myc in many human cancers.

  1. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

    Directory of Open Access Journals (Sweden)

    Brigitte Mack

    Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

  2. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Science.gov (United States)

    Kadohama, Noriaki; Goh, Tatsuaki; Ohnishi, Miwa; Fukaki, Hidehiro; Mimura, Tetsuro; Suzuki, Yoshihiro

    2013-01-01

    It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg') and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  3. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Directory of Open Access Journals (Sweden)

    Noriaki Kadohama

    Full Text Available It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg' and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  4. Microfluidic device for rapid solution exchange to study kinetics of cell physiology

    Science.gov (United States)

    Hu, Howard; Honnatti, Meghana; Gillis, Kevin

    2006-11-01

    Exchanging the extracellular solution of the cell rapidly (less than 10ms) is an important requirement in study the kinetics of cell physiology. A microfluidic device is developed to exchange the solution around the cells as they flow through a junction at the intersection of two microfluidic channels. The solution exchange time is measured experimentally by fluorescently labeling the cell surface membranes with a styryl dye, FM1-43 or FM 2-10, and then observing the time course of cell fluorescence decay following the rapid drop in the extracellular concentration of the FM dye that occurs as the cell flows past the fluidic junction. A numerical model is developed to guide the experimental design of microfluidic device. In the model, the motion of a single cell through a fluid junction is simulated and the mixing process of the solutions is solved. The model also includes the kinetics of departitioning of FM dyes from the cell membrane. The departitioning time constants for the FM dyes are determined from fitting the measured data of the cell fluorescence decay. This departitioning kinetics is important as FM dyes are commonly used to label cell membranes for the purpose of measuring the release of neurotransmitter from synaptic vesicles via exocytosis and the subsequent reuptake of vesicular membrane by endocytosis.

  5. Rapid characterization of the biomechanical properties of drug-treated cells in a microfluidic device

    Science.gov (United States)

    Zhang, Xiaofei; Chu, Henry K.; Zhang, Yang; Bai, Guohua; Wang, Kaiqun; Tan, Qiulin; Sun, Dong

    2015-10-01

    Cell mechanics is closely related to many cell functions. Recent studies have suggested that the deformability of cells can be an effective biomarker to indicate the onset and progression of diseases. In this paper, a microfluidic chip is designed for rapid characterization of the mechanics of drug-treated cells through stretching with dielectrophoresis (DEP) force. This chip was fabricated using PDMS and micro-electrodes were integrated and patterned on the ITO layer of the chip. Leukemia NB4 cells were considered and the effect of all-trans retinoic acid (ATRA) drug on NB4 cells were examined via the microfluidic chip. To induce a DEP force onto the cell, a relatively weak ac voltage was utilized to immobilize a cell at one side of the electrodes. The applied voltage was then increased to 3.5 V pp and the cell started to be stretched along the applied electric field lines. The elongation of the cell was observed using an optical microscope and the results showed that both types of cells were deformed by the induced DEP force. The strain of the NB4 cell without the drug treatment was recorded to be about 0.08 (time t = 180 s) and the drug-treated NB4 cell was about 0.21 (time t = 180 s), indicating a decrease in the stiffness after drug treatment. The elastic modulus of the cell was also evaluated and the modulus changed from 140 Pa to 41 Pa after drug treatment. This microfluidic chip can provide a simple and rapid platform for measuring the change in the biomechanical properties of cells and can potentially be used as the tool to determine the biomechanical effects of different drug treatments for drug discovery and development applications.

  6. A bacteriophage endolysin-based electrochemical impedance biosensor for the rapid detection of Listeria cells.

    Science.gov (United States)

    Tolba, Mona; Ahmed, Minhaz Uddin; Tlili, Chaker; Eichenseher, Fritz; Loessner, Martin J; Zourob, Mohammed

    2012-12-21

    The objective of this study was to develop a biosensor using the cell wall binding domain (CBD) of bacteriophage-encoded peptidoglycan hydrolases (endolysin) immobilized on a gold screen printed electrode (SPE) and subsequent electrochemical impedance spectroscopy (EIS) for a rapid and specific detection of Listeria cells. The endolysin was amine-coupled to SPEs using EDC/NHS chemistry. The CBD-based electrode was used to capture and detect the Listeria innocua serovar 6b from pure culture and 2% artificially contaminated milk. In our study, the endolysin functionalized SPEs have been characterized using X-ray photoelectron spectroscopy (XPS). The integration of endolysin-based recognition for specific bacteria and EIS can be used for direct and rapid detection of Listeria cells with high specificity against non-Listeria cells with a limit of detection of 1.1 × 10(4) and 10(5) CFU mL(-1) in pure culture and 2% milk, respectively.

  7. A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells.

    Directory of Open Access Journals (Sweden)

    Victor Mauri

    Full Text Available A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG, in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB, a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in

  8. Rapidly acquired cytotoxicity of lymphoid cells from ice inoculated with allogeneic spleen cells.

    Science.gov (United States)

    Slavina, E G; Karmanova, N V; Leipunskaya, I L; Zinzar, S N; Reinhöfer, J; Svet-Moldavsky, G J

    1976-12-01

    Spleen cells from C57BL/6J or CBA mice inoculated iv with spleen cells from BALB/c mice produced a strong nonspecific cytotoxic effect on target cells (mouse L-cells). Lymph node cells from CBA or C57BL/6J mice inoculated sc with BALB/c spleen cells also destroyed L-cells. Lymph node cells from mice inoculated with syngeneic spleen cells were not cytotoxic. The cytotoxic effect was observed ion of allogeneic but not syngeneic spleen cells. This effect was considerably reduced or completely suppressed after partial or total removal of plastic-adherent cells.

  9. Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

    Science.gov (United States)

    Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

    2015-01-01

    We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

  10. Rapid Evaluation of Power Degradation in Series Connection of Single Feeding Microsized Microbial Fuel Cells

    KAUST Repository

    Rojas, Jhonathan Prieto

    2014-07-08

    We have developed a sustainable, single feeding, microsized, air-cathode and membrane-free microbial fuel cells with a volume of 40 mu L each, which we have used for rapid evaluation of power generation and viability of a series array of three cells seeking higher voltage levels. Contrary to expectations, the achieved power density was modest (45 mWm(-3)), limited due to non-uniformities in assembly and the single-channel feeding system.

  11. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    Directory of Open Access Journals (Sweden)

    Wenfeng Liang

    Full Text Available Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell sample from red blood cells (RBCs with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for

  12. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    Science.gov (United States)

    Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing

    2014-01-01

    Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and

  13. Rapid reactivation of extralymphoid CD4 T cells during secondary infection.

    Directory of Open Access Journals (Sweden)

    Timothy J Chapman

    Full Text Available After infection, extralymphoid tissues are enriched with effector and memory T cells of a highly activated phenotype. The capacity for rapid effector cytokine response from extralymphoid tissue-memory T cells suggests these cells may perform a 'sentinel' function in the tissue. While it has been demonstrated that extralymphoid CD4+ T cells can directly respond to secondary infection, little is known about how rapidly this response is initiated, and how early activation of T cells in the tissue may affect the innate response to infection. Here we use a mouse model of secondary heterosubtypic influenza infection to show that CD4(+ T cells in the lung airways are reactivated within 24 hours of secondary challenge. Airway CD4(+ T cells initiate an inflammatory cytokine and chemokine program that both alters the composition of the early innate response and contributes to the reduction of viral titers in the lung. These results show that, unlike a primary infection, extralymphoid tissue-memory CD4(+ T cells respond alongside the innate response during secondary infection, thereby shaping the overall immune profile in the airways. These data provide new insights into the role of extralymphoid CD4(+ T cells during secondary immune responses.

  14. Transcriptional profiling of ParA and ParB mutants in actively dividing cells of an opportunistic human pathogen Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Aneta A Bartosik

    Full Text Available Accurate chromosome segregation to progeny cells is a fundamental process ensuring proper inheritance of genetic material. In bacteria with simple cell cycle, chromosome segregation follows replication initiation since duplicated oriC domains start segregating to opposite halves of the cell soon after they are made. ParA and ParB proteins together with specific DNA sequences are parts of the segregation machinery. ParA and ParB proteins in Pseudomonas aeruginosa are important for optimal growth, nucleoid segregation, cell division and motility. Comparative transcriptome analysis of parA null and parB null mutants versus parental P. aeruginosa PAO1161 strain demonstrated global changes in gene expression pattern in logarithmically growing planktonic cultures. The set of genes similarly affected in both mutant strains is designated Par regulon and comprises 536 genes. The Par regulon includes genes controlled by two sigma factors (RpoN and PvdS as well as known and putative transcriptional regulators. In the absence of Par proteins, a large number of genes from RpoS regulon is induced, reflecting the need for slowing down the cell growth rate and decelerating the metabolic processes. Changes in the expression profiles of genes involved in c-di-GMP turnover point out the role of this effector in such signal transmission. Microarray data for chosen genes were confirmed by RT-qPCR analysis. The promoter regions of selected genes were cloned upstream of the promoter-less lacZ gene and analyzed in the heterologous host E. coliΔlac. Regulation by ParA and ParB of P. aeruginosa was confirmed for some of the tested promoters. Our data demonstrate that ParA and ParB besides their role in accurate chromosome segregation may act as modulators of genes expression. Directly or indirectly, Par proteins are part of the wider regulatory network in P. aeruginosa linking the process of chromosome segregation with the cell growth, division and motility.

  15. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

    Science.gov (United States)

    Takahashi, Tadanobu; Agarikuchi, Takashi; Kurebayashi, Yuuki; Shibahara, Nona; Suzuki, Chihiro; Kishikawa, Akiko; Fukushima, Keijo; Takano, Maiko; Suzuki, Fumie; Wada, Hirohisa; Otsubo, Tadamune; Ikeda, Kiyoshi; Minami, Akira; Suzuki, Takashi

    2015-01-01

    Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  16. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

    Directory of Open Access Journals (Sweden)

    Tadanobu Takahashi

    Full Text Available Mumps viruses show diverse cytopathic effects (CPEs of infected cells and viral plaque formation (no CPE or no plaque formation in some cases depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac, was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study, even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  17. Cost-effective and rapid blood analysis on a cell-phone.

    Science.gov (United States)

    Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan

    2013-04-07

    We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

  18. miRNA expression profiling divides follicular dendritic cell sarcomas into two groups, related to fibroblasts and myopericytomas or Castleman's disease.

    Science.gov (United States)

    Hartmann, Sylvia; Döring, Claudia; Agostinelli, Claudio; Portscher-Kim, Soo-Jeong; Lonardi, Silvia; Lorenzi, Luisa; Fuligni, Fabio; Martinez, Daniel; Mehta, Jay; Borges, Anita; Hackstein, Holger; Kippenberger, Stefan; Piccaluga, Pier Paolo; Simonitsch-Klupp, Ingrid; Cabeçadas, José; Campo, Elias; Facchetti, Fabio; Pileri, Stefano A; Hansmann, Martin-Leo

    2016-09-01

    Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumours, which are fatal in 20% of the patients and usually occur in secondary lymphoid organs or extranodal localizations. Due to the rareness of these tumours, only few studies have been conducted on molecular level. In the present study, we performed microRNA (miRNA) profiling of 31 FDC sarcomas and identified two subgroups, one with high miRNA expression and the other group with low miRNA expression levels. The first group showed a strong similarity to fibroblasts and myopericytomas, whereas the second group was more closely related to FDCs from Castleman's disease. Both groups showed important differences compared with myeloid-derived dendritic cells, confirming mesenchymal origin of FDCs and their derived sarcomas. The two FDC sarcoma groups did not differ on morphological grounds, mitotic activity or BRAF mutation status. However, patients of group I presented a tendency to a shorter overall survival and more frequent podoplanin expression by immunohistochemistry. The importance of these newly recognized FDC sarcoma subgroups in terms of clinical behaviour and therapeutic implications should be assessed in a larger cohort in future studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. The acoustic sensor for rapid analysis of bacterial cells in the conductive suspensions.

    Science.gov (United States)

    Borodina, I A; Zaitsev, B D; Guliy, O; Teplykh, A A; Shikhabudinov, A M

    2017-11-01

    The possibility of using the acoustic sensor on the basis of a two-channel delay line for rapid analysis of bacterial cells in the conductive suspensions was investigated. The dependencies of change in phase and insertion loss of output signal of the sensor on conductivity of buffer solution with various concentrations of cells due to a specific interaction "bacterial cells - mini-antibodies" for electrically open and electrically shorted channels of delay line were measured. It has been found that these changes have the most values for the electrically open channel. It has been also shown that the sensor rapidly responds to the specific interaction and the time stabilization of the phase and insertion loss of output signal is less than 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Jian Ning

    2011-01-01

    Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

  1. B-Cell Depletion Salvage Therapy in Rapidly Progressive Dermatomyositis Related Interstitial Lung Disease.

    Science.gov (United States)

    Eissa, Khaled; Palomino, Jaime

    2016-01-01

    Interstitial lung disease (ILD) is a major cause of morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). Glucocorticoids are the initial standard treatment. However, many patients fail to respond and continue to progress despite treatment with high dose glucocorticoids. The efficacy of rituximab has been suggested in case reports and case series of refractory antisynthetase (AS) syndrome, but data on patients without auto-antibodies or with rapidly progressive ILD are scarce. We report a case of rapidly progressive dermatomyositis (DM) associated ILD treated successfully with B-cell depletion therapy.

  2. Cell-scale dynamic recycling and cortical flow of the actin–myosin cytoskeleton for rapid cell migration

    Directory of Open Access Journals (Sweden)

    Shigehiko Yumura

    2012-11-01

    Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton.

  3. Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

    KAUST Repository

    Maruyama, Daisuke

    2015-05-01

    In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization. Two female gametes (the egg cell and the central cell) in flowering plants coordinately prevent attractions of excess number of pollen tubes via two mechanisms to inactivate persistent synergid cell. © 2015 Elsevier Inc.

  4. Rapid morphological oscillation of mitochondrion-rich cell in estuarine mudskipper following salinity changes.

    Science.gov (United States)

    Sakamoto, T; Yokota, S; Ando, M

    2000-05-01

    Morphological changes in the chloride cells or mitochondrion-rich (MR) cells in the skin under the pectoral fin of the estuarine mudskipper (Periophthalmus modestus) were examined in relation to intertidal salinity oscillation in river mouth. MR cells were distinguished between those in contact with the water (cells labeled with both mitochondrial probe DASPEI and Concanavalin-A, an apical surface marker of MR cells) and those that are not (DASPEI-positive only). After transfer of the fish from seawater to freshwater, no difference in the total MR cell density was observed, but the subpopulation of MR cells that are Concanavalin-A-positive decreased dramatically within 30 min. After 6 hr in freshwater, the fish were returned to seawater; the number of Con-A-positive MR cells increased to the initial levels rapidly. Thus, in seawater, mudskippers seem to open the apical crypts of the MR cells to secrete salt; in freshwater, they close the crypt of the MR cells tentatively, and tolerate hypotonicity until the rising tide. This unique response of chloride cells may also be seen in gills of other estuarine species.

  5. Hyaluronic Acid-Serum Hydrogels Rapidly Restore Metabolism of Encapsulated Stem Cells and Promote Engraftment

    Science.gov (United States)

    Chan, Angel T.; Karakas, Mehmet F.; Vakrou, Styliani; Afzal, Junaid; Rittenbach, Andrew; Lin, Xiaoping; Wahl, Richard L.; Pomper, Martin G.; Steenbergen, Charles J.; Tsui, Benjamin M.W.; Elisseeff, Jennifer H.; Abraham, M. Roselle

    2015-01-01

    Background Cell death due to anoikis, necrosis and cell egress from transplantation sites limits functional benefits of cellular cardiomyoplasty. Cell dissociation and suspension, which are a pre-requisite for most cell transplantation studies, lead to depression of cellular metabolism and anoikis, which contribute to low engraftment. Objective We tissue engineered scaffolds with the goal of rapidly restoring metabolism, promoting viability, proliferation and engraftment of encapsulated stem cells. Methods The carboxyl groups of HA were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA-NHS) groups that react with free amine groups to form amide bonds. HA-NHS was cross-linked by serum to generate HA:Serum (HA:Ser) hydrogels. Physical properties of HA:Ser hydrogels were measured. Effect of encapsulating cardiosphere-derived cells (CDCs) in HA:Ser hydrogels on viability, proliferation, glucose uptake and metabolism was assessed in vitro. In vivo acute intra-myocardial cell retention of 18FDG-labeled CDCs encapsulated in HA:Ser hydrogels was quantified. Effect of CDC encapsulation in HA:Ser hydrogels on in vivo metabolism and engraftment at 7 days was assessed by serial, dual isotope SPECT-CT and bioluminescence imaging of CDCs expressing the Na-iodide symporter and firefly luciferase genes respectively. Effect of HA:Ser hydrogels +/− CDCs on cardiac function was assessed at 7 days & 28 days post-infarct. Results HA:Ser hydrogels are highly bio-adhesive, biodegradable, promote rapid cell adhesion, glucose uptake and restore bioenergetics of encapsulated cells within 1 h of encapsulation, both in vitro and in vivo. These metabolic scaffolds can be applied epicardially as a patch to beating hearts or injected intramyocardially. HA:Ser hydrogels markedly increase acute intramyocardial retention (~6 fold), promote in vivo viability, proliferation, engraftment of encapsulated stem cells and angiogenesis. Conclusion HA:Ser hydrogels

  6. Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy.

    Science.gov (United States)

    Li, Mi; Xiao, Xiubin; Liu, Lianqing; Xi, Ning; Wang, Yuechao

    2016-09-01

    Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin-biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20-rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in

  7. Dividing Fractions: A Pedagogical Technique

    Science.gov (United States)

    Lewis, Robert

    2016-01-01

    When dividing one fraction by a second fraction, invert, that is, flip the second fraction, then multiply it by the first fraction. To multiply fractions, simply multiply across the denominators, and multiply across the numerators to get the resultant fraction. So by inverting the division of fractions it is turned into an easy multiplication of…

  8. Dividing Attention Increases Operational Momentum

    Directory of Open Access Journals (Sweden)

    Koleen McCrink

    2017-12-01

    Full Text Available When adding or subtracting two quantities, adults often compute an estimated outcome that is larger or smaller, respectively, than the actual outcome, a bias referred to as “operational momentum”. The effects of attention on operational momentum were investigated. Participants viewed a display in which two arrays of objects were added, or one array was subtracted from another array, and judged whether a subsequent outcome (probe array contained the correct or incorrect number of objects. In a baseline condition, only the arrays to be added or subtracted were viewed. In divided attention conditions, participants simultaneously viewed a sequence of colors or shapes, and judged which color (a non-spatial judgment or shape (a spatial judgment was repeated. Operational momentum occurred in all conditions, but was higher in divided attention conditions than in the baseline condition, primarily for addition problems. This pattern suggests that dividing attention, rather than decreasing operational momentum by decreasing attentional shifts, actually increased operational momentum. These results are consistent with a heightened use of arithmetic heuristics under conditions of divided attention.

  9. Rapid, efficient charging of lead-acid and nickel-zinc traction cells. [for electric vehicles

    Science.gov (United States)

    Smithrick, J. J.

    1978-01-01

    Lead-acid and nickel-zinc traction cells were rapidly and efficiently charged using a high rate taped dc charge (HRTDC) method which could possibly be used for on-the-road service recharge of electric vehicles. The HRTDC method takes advantage of initial high cell charge acceptance and uses cell gassing rate and temperature as an indicator of charging efficiency. On the average, 300 amp-hour nickel-zinc traction cells were given a HRTDC to 78% of rated amp-hour capacity within 53 minutes at an amp-hour efficiency of 92% and an energy efficiency of 52%. Three-hundred amp-hour lead-acid traction cells were charged to 69% of rated amp-hour capacity within 46 minutes at an amp-hour efficiency of 91% with an energy efficiency of 64%.

  10. Optical scatter imaging: a microscopic modality for the rapid morphological assay of living cells

    Science.gov (United States)

    Boustany, Nada N.

    2007-02-01

    Tumors derived from epithelial cells comprise the majority of human tumors and their growth results from the accumulation of multiple mutations affecting cellular processes critical for tissue homeostasis, including cell proliferation and cell death. To understand these processes and address the complexity of cancer cell function, multiple cellular responses to different experimental conditions and specific genetic mutations must be analyzed. Fundamental to this endeavor is the development of rapid cellular assays in genetically defined cells, and in particular, the development of optical imaging methods that allow dynamic observation and real-time monitoring of cellular processes. In this context, we are developing an optical scatter imaging technology that is intended to bridge the gap between light and electron microscopy by rapidly providing morphometric information about the relative size and shape of non-spherical organelles, with sub-wavelength resolution. Our goal is to complement current microscopy techniques used to study cells in-vitro, especially in long-term time-lapse studies of living cells, where exogenous labels can be toxic, and electron microscopy will destroy the sample. The optical measurements are based on Fourier spatial filtering in a standard microscope, and could ultimately be incorporated into existing high-throughput diagnostic platforms for cancer cell research and histopathology of neoplastic tissue arrays. Using an engineered epithelial cell model of tumor formation, we are currently studying how organelle structure and function are altered by defined genetic mutations affecting the propensity for cell death and oncogenic potential, and by environmental conditions promoting tumor growth. This talk will describe our optical scatter imaging technology and present results from our studies on apoptosis, and the function of BCL-2 family proteins.

  11. Rapid and serial quantification of adhesion forces of yeast and Mammalian cells.

    Directory of Open Access Journals (Sweden)

    Eva Potthoff

    Full Text Available Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM. In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.

  12. Advances in a rapidly emerging field of hair follicle stem cell research.

    Science.gov (United States)

    Mokos, Zrinka Bukvić; Mosler, Elvira Lazić

    2014-03-01

    Human skin maintains the ability to regenerate during adulthood, as it constantly renews itself throughout adult life, and the hair follicle (HF) undergoes a perpetual cycle of growth and degeneration. The study of stem cells (SCs) in the epidermis and skin tissue engineering is a rapidly emerging field, where advances have been made in both basic and clinical research. Advances in basic science include the ability to assay SCs of the epidermis in vivo, identification of an independent interfollicular epidermal SC, and improved ability to analyze individual SCs divisions, as well as the recent hair organ regeneration via the bioengineered hair follicular unit transplantation (FUT) in mice. Advances in the clinic include recognition of the importance of SCs for wound repair and for gene therapy in inherited skin diseases, for example epidermolysis bullosa. The study of the HF stem cells (HFSCs) started by identification of epidermal SC in the HF bulge as quiescent "label retaining cells". The research of these cells emerged rapidly after the identification of bulge cell molecular markers, such as keratin 15 (K15) and CD34 in mice and CD200 in humans, which allowed the isolation and characterization of bulge cells from follicles. This paper provides an overview of the current knowledge on epidermal SCs in the HF describing their essential characteristics and the control of follicle SCs fate, their role in alopecia, as well as their use in tissue engineering.

  13. Digital Divide and Social Media: Connectivity Doesn’t End the Digital Divide, Skills Do

    OpenAIRE

    Radovanovic, Danica

    2011-01-01

    Whether we like it or not, we live in a very unequal and stratified world. We live in societies in which inequality is ignored in education, science, and in the social media. As Internet technologies are rapidly evolving and new digital divides on the Internet emerge, we must move beyond, at some point, a singular concern over Internet access and technological infrastructure issues. We must tackle socio-cultural differences, we must focus on Internet skills, literacies and social media usage.

  14. Rapid metabolism of exogenous angiotensin II by catecholaminergic neuronal cells in culture media.

    Science.gov (United States)

    Basu, Urmi; Seravalli, Javier; Madayiputhiya, Nandakumar; Adamec, Jiri; Case, Adam J; Zimmerman, Matthew C

    2015-02-01

    Angiotensin II (AngII) acts on central neurons to increase neuronal firing and induce sympathoexcitation, which contribute to the pathogenesis of cardiovascular diseases including hypertension and heart failure. Numerous studies have examined the precise AngII-induced intraneuronal signaling mechanism in an attempt to identify new therapeutic targets for these diseases. Considering the technical challenges in studying specific intraneuronal signaling pathways in vivo, especially in the cardiovascular control brain regions, most studies have relied on neuronal cell culture models. However, there are numerous limitations in using cell culture models to study AngII intraneuronal signaling, including the lack of evidence indicating the stability of AngII in culture media. Herein, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Using liquid chromatography-tandem mass spectrometry, we measured levels of AngII and its metabolites, Ang III, Ang IV, and Ang-1-7, in neuronal cell culture media after administration of exogenous AngII (100 nmol/L) to a neuronal cell culture model (CATH.a neurons). AngII levels rapidly declined in the media, returning to near baseline levels within 3 h of administration. Additionally, levels of Ang III and Ang-1-7 acutely increased, while levels of Ang IV remained unchanged. Replenishing the media with exogenous AngII every 3 h for 24 h resulted in a consistent and significant increase in AngII levels for the duration of the treatment period. These data indicate that AngII is rapidly metabolized in neuronal cell culture media, and replenishing the media at least every 3 h is needed to sustain chronically elevated levels. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  15. Dexamethasone rapidly suppresses IL-33-stimulated mast cell function by blocking transcription factor activity.

    Science.gov (United States)

    Paranjape, Anuya; Chernushevich, Oksana; Qayum, Amina Abdul; Spence, Andrew J; Taruselli, Marcela T; Abebayehu, Daniel; Barnstein, Brian O; McLeod, Jamie Josephine Avila; Baker, Bianca; Bajaj, Gurjas S; Chumanevich, Alena P; Oskeritzian, Carole A; Ryan, John J

    2016-12-01

    Mast cells are critical effectors of allergic disease and can be activated by IL-33, a proinflammatory member of the IL-1 cytokine family. IL-33 worsens the pathology of mast cell-mediated diseases, but therapies to antagonize IL-33 are still forthcoming. Because steroids are the mainstay of allergic disease treatment and are well known to suppress mast cell activation by other stimuli, we examined the effects of the steroid dexamethasone on IL-33-mediated mast cell function. We found that dexamethasone potently and rapidly suppressed cytokine production elicited by IL-33 from murine bone marrow-derived and peritoneal mast cells. IL-33 enhances IgE-mediated mast cell cytokine production, an activity that was also antagonized by dexamethasone. These effects were consistent in human mast cells. We additionally observed that IL-33 augmented migration of IgE-sensitized mast cells toward antigen. This enhancing effect was similarly reversed by dexamethasone. Simultaneous addition of dexamethasone with IL-33 had no effect on the phosphorylation of MAP kinases or NFκB p65 subunit; however, dexamethasone antagonized AP-1- and NFκB-mediated transcriptional activity. Intraperitoneal administration of dexamethasone completely abrogated IL-33-mediated peritoneal neutrophil recruitment and prevented plasma IL-6 elevation. These data demonstrate that steroid therapy may be an effective means of antagonizing the effects of IL-33 on mast cells in vitro and in vivo, acting partly by suppressing IL-33-induced NFκB and AP-1 activity. © Society for Leukocyte Biology.

  16. Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection.

    Directory of Open Access Journals (Sweden)

    William H Thiel

    Full Text Available The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX with high-throughput sequencing (HTS and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs. Several rounds of positive (VSMCs and negative (endothelial cells; ECs selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1 metrics of selection enrichment; and (2 pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

  17. Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection.

    Science.gov (United States)

    Thiel, William H; Bair, Thomas; Peek, Andrew S; Liu, Xiuying; Dassie, Justin; Stockdale, Katie R; Behlke, Mark A; Miller, Francis J; Giangrande, Paloma H

    2012-01-01

    The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers. We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs. We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

  18. Rapid Fatal Outcome from Pulmonary Arteries Compression in Transitional Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Ioannis A. Voutsadakis

    2009-01-01

    Full Text Available Transitional cell carcinoma of the urinary bladder is a malignancy that metastasizes frequently to lymph nodes including the mediastinal lymph nodes. This occurrence may produce symptoms due to compression of adjacent structures such as the superior vena cava syndrome or dysphagia from esophageal compression. We report the case of a 59-year-old man with metastatic transitional cell carcinoma for whom mediastinal lymphadenopathy led to pulmonary artery compression and a rapidly fatal outcome. This rare occurrence has to be distinguished from pulmonary embolism, a much more frequent event in cancer patients, in order that proper and prompt treatment be initiated.

  19. Bridging the Health Data Divide.

    Science.gov (United States)

    Celi, Leo Anthony; Davidzon, Guido; Johnson, Alistair Ew; Komorowski, Matthieu; Marshall, Dominic C; Nair, Sunil S; Phillips, Colin T; Pollard, Tom J; Raffa, Jesse D; Salciccioli, Justin D; Salgueiro, Francisco Muge; Stone, David J

    2016-12-20

    Fundamental quality, safety, and cost problems have not been resolved by the increasing digitization of health care. This digitization has progressed alongside the presence of a persistent divide between clinicians, the domain experts, and the technical experts, such as data scientists. The disconnect between clinicians and data scientists translates into a waste of research and health care resources, slow uptake of innovations, and poorer outcomes than are desirable and achievable. The divide can be narrowed by creating a culture of collaboration between these two disciplines, exemplified by events such as datathons. However, in order to more fully and meaningfully bridge the divide, the infrastructure of medical education, publication, and funding processes must evolve to support and enhance a learning health care system. ©Leo Anthony Celi, Guido Davidzon, Alistair EW Johnson, Matthieu Komorowski, Dominic C Marshall, Sunil S Nair, Colin T Phillips, Tom J Pollard, Jesse D Raffa, Justin D Salciccioli, Francisco Muge Salgueiro, David J Stone. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 20.12.2016.

  20. Divided by the Market, Divided by the State

    DEFF Research Database (Denmark)

    Wulfgramm, Melike; Starke, Peter

    2017-01-01

    of the individual differences, the analysis also shows, for the first time, that both high inequality and strongly redistributive policies divide public opinion along the lines of socioeconomic position. Put differently, while market inequality may be associated with less cohesive attitudes, a highly redistributive...... welfare state does not seem to foster agreement among the public, either. These findings have important policy implications for advanced welfare states, including a renewed emphasis on ‘predistribution’ (i.e., policies that influence the primary distribution of income) in order to avoid the scenario...

  1. Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41.

    Science.gov (United States)

    Vervelde, L; Matthijs, M G R; van Haarlem, D A; de Wit, J J; Jansen, C A

    2013-02-15

    Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Migratory dermal dendritic cells act as rapid sensors of protozoan parasites.

    Directory of Open Access Journals (Sweden)

    Lai Guan Ng

    2008-11-01

    Full Text Available Dendritic cells (DC, including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Galpha(i protein-coupled receptor-dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.

  3. Rapid deterioration of preexisting renal insufficiency after autologous mesenchymal stem cell therapy

    Directory of Open Access Journals (Sweden)

    Jun-Seop Kim

    2017-06-01

    Full Text Available Administration of autologous mesenchymal stem cells (MSCs has been shown to improve renal function and histological findings in acute kidney injury (AKI models. However, its effects in chronic kidney disease (CKD are unclear, particularly in the clinical setting. Here, we report our experience with a CKD patient who was treated by intravenous infusion of autologous MSCs derived from adipose tissue in an unknown clinic outside of Korea. The renal function of the patient had been stable for several years before MSC administration. One week after the autologous MSC infusion, the preexisting renal insufficiency was rapidly aggravated without any other evidence of AKI. Hemodialysis was started 3 months after MSC administration. Renal biopsy findings at dialysis showed severe interstitial fibrosis and inflammatory cell infiltration, with a few cells expressing CD34 and CD117, 2 surface markers of stem cells. This case highlights the potential nephrotoxicity of autologous MSC therapy in CKD patients.

  4. Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

    Directory of Open Access Journals (Sweden)

    Sarah E Kelly

    2010-04-01

    Full Text Available Cancer stem cells (CSCs are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB (Celdyne, Houston, TX. For comparison, another bioreactor, the rotary cell culture system (RCCS manufactured by Synthecon (Houston, TX was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

  5. Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology

    Energy Technology Data Exchange (ETDEWEB)

    Sundaram, S. K.; Sacksteder, Colette A.; Weber, T. J.; Riley, Brian J.; Addleman, Raymond S.; Harrer, Bruce J.; Peterman, John W.

    2013-01-01

    A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live-cells interact with an external stimulus, e.g., a nanosized particle (NSP), and the toxicity and broad risk associated with these stimuli. NSPs are increasingly used in medicine with largely undetermined hazards in complex cell dynamics and environments. It is difficult to capture the complexity and dynamics of these interactions by following an omics-based approach exclusively, which are expensive and time-consuming. Additionally, this approach needs destructive sampling methods. Live-cell attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry is well suited to provide noninvasive approach to provide rapid screening of cellular responses to potentially toxic NSPs or any stimuli. Herein we review the technical basis of the approach, the instrument configuration and interface with the biological media, and various effects that impact the data, data analysis, and toxicity. Our preliminary results on live-cell monitoring show promise for rapid screening the NSPs.

  6. Rapid in vitro derivation of endothelium directly from human cancer cells.

    Directory of Open Access Journals (Sweden)

    Jennifer D Elster

    Full Text Available The development of an independent blood supply by a tumor is essential for maintaining growth beyond a certain limited size and for providing a portal for metastatic dissemination. Host-derived endothelial cells (ECs residing in and compromising the tumor vasculature originate via distinct processes known as sprouting angiogenesis and vasculogenesis. More recently ECs originating directly from the tumor cells themselves have been described although the basis for this phenomenon remains poorly understood. Here we describe in vitro conditions that allow lung and ovarian cancer cells to undergo a rapid and efficient transition into ECs that are indistinguishable from those obtained in vivo. A variety of methods were used to establish that the acquired phenotypes and behaviors of these tumor-derived ECs (TDECs closely resemble those of authentic ECs. Xenografts arising from co-inoculated in vitro-derived TDECs and tumor cells were also more highly vascularized than control tumors; moreover, their blood vessels were on average larger and frequently contained admixtures of host-derived ECs and TDECs derived from the initial inoculum. These results demonstrate that cancer cells can be manipulated under well-defined in vitro conditions to initiate a tumor cell-to-EC transition that is largely cell-autonomous, highly efficient and closely mimics the in vivo process. These studies provide a suitable means by which to identify and perhaps modify the earliest steps in TDEC generation.

  7. Rapid Prototyping of Polymeric Nanopillars by 3D Direct Laser Writing for Controlling Cell Behavior.

    Science.gov (United States)

    Buch-Månson, Nina; Spangenberg, Arnaud; Gomez, Laura Piedad Chia; Malval, Jean-Pierre; Soppera, Olivier; Martinez, Karen L

    2017-08-23

    Mammalian cells have been widely shown to respond to nano- and microtopography that mimics the extracellular matrix. Synthetic nano- and micron-sized structures are therefore of great interest in the field of tissue engineering, where polymers are particularly attractive due to excellent biocompatibility and versatile fabrication methods. Ordered arrays of polymeric pillars provide a controlled topographical environment to study and manipulate cells, but processing methods are typically either optimized for the nano- or microscale. Here, we demonstrate polymeric nanopillar (NP) fabrication using 3D direct laser writing (3D DLW), which offers a rapid prototyping across both size regimes. The NPs are interfaced with NIH3T3 cells and the effect of tuning geometrical parameters of the NP array is investigated. Cells are found to adhere on a wide range of geometries, but the interface depends on NP density and length. The Cell Interface with Nanostructure Arrays (CINA) model is successfully extended to predict the type of interface formed on different NP geometries, which is found to correlate with the efficiency of cell alignment along the NPs. The combination of the CINA model with the highly versatile 3D DLW fabrication thus holds the promise of improved design of polymeric NP arrays for controlling cell growth.

  8. Overcoming challenges to initiating cell therapy clinical trials in rapidly developing countries: India as a model.

    Science.gov (United States)

    Viswanathan, Sowmya; Rao, Mahendra; Keating, Armand; Srivastava, Alok

    2013-08-01

    Increasingly, a number of rapidly developing countries, including India, China, Brazil, and others, are becoming global hot spots for the development of regenerative medicine applications, including stem cell-based therapies. Identifying and overcoming regulatory and translational research challenges and promoting scientific and ethical clinical trials with cells will help curb the growth of stem cell tourism for unproven therapies. It will also enable academic investigators, local regulators, and national and international biotechnology and biopharmaceutical companies to accelerate stem cell-based clinical research that could lead to effective innovative treatments in these regions. Using India as a model system and obtaining input from regulators, clinicians, academics, and industry representatives across the stem cell field in India, we reviewed the role of key agencies and processes involved in this field. We have identified areas that need attention and here provide solutions from other established and functioning models in the world to streamline and unify the regulatory and ethics approval processes for cell-based therapies. We also make recommendations to check the growth and functioning of clinics offering unproven treatments. Addressing these issues will remove considerable hurdles to both local and international investigators, accelerate the pace of research and development, and create a quality environment for reliable products to emerge. By doing so, these countries would have taken one important step to move to the forefront of stem cell-based therapeutics.

  9. Simple and Rapid Bioink Jet Printing for Multiscale Cell Adhesion Islands.

    Science.gov (United States)

    Mecozzi, Laura; Gennari, Oriella; Rega, Romina; Battista, Luigi; Ferraro, Pietro; Grilli, Simonetta

    2017-03-01

    A simple and rapid process for multiscale printing of bioinks with dot widths ranging from hundreds of microns down to 0.5 μm is presented. The process makes use of spontaneous surface charges generated pyroelectrically that are able to draw little daughter droplets directly from the free meniscus of a mother drop through jetting ("p-jet"), thus avoiding time-consuming and expensive fabrication of microstructured nozzles. Multiscale can be easily achieved by modulating the parameters of the p-jet process. Here, it is shown that the p-jet allows us to print well-defined adhesion islands where NIH-3T3 fibroblasts are constrained to live into cluster configurations ranging from 20 down to single cell level. The proposed fabrication approach can be useful for high-throughput studies on cell adhesion, cytoskeleton organization, and stem cell differentiation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Short communication: HIV type 1 escapes inactivation by saliva via rapid escape into oral epithelial cells.

    Science.gov (United States)

    Dietrich, Elizabeth A; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Kappes, John C; Ross, Karen F; Herzberg, Mark C

    2012-12-01

    Saliva contains anti-HIV-1 factors, which show unclear efficacy in thwarting mucosal infection. When incubated in fresh, unfractionated whole saliva, infectious HIV-1 IIIb and BaL (X4- and R5-tropic, respectively) persisted from 4 to at least 30 min in a saliva concentration-dependent manner. In salivary supernatant for up to 6 h, both infectious HIV-1 strains "escaped" into immortalized oral epithelial cells; infectious BaL showed selectively enhanced escape in the presence of saliva. Fluorescently labeled HIV-1 virus-like particles entered oral epithelial cells within minutes of exposure. Using a previously unrecognized mechanism, therefore, strains of HIV-1 escape inactivation by saliva via rapid uptake into oral epithelial cells.

  11. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy

    Directory of Open Access Journals (Sweden)

    Wang Xueying

    2008-10-01

    Full Text Available Abstract Background Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed. Results The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion. Conclusion New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality.

  12. Random migration and signal integration promote rapid and robust T cell recruitment.

    Science.gov (United States)

    Textor, Johannes; Henrickson, Sarah E; Mandl, Judith N; von Andrian, Ulrich H; Westermann, Jürgen; de Boer, Rob J; Beltman, Joost B

    2014-08-01

    To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance.

  13. Rapid spread of mouse mammary tumor virus in cultured human breast cells

    Directory of Open Access Journals (Sweden)

    Günzburg Walter H

    2007-10-01

    Full Text Available Abstract Background The role of mouse mammary tumor virus (MMTV as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection. Results Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR, in cultured human mammary cells (Hs578T, ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences. Conclusion Taken together, our results show that human cells can support replication of mouse mammary tumor virus.

  14. Primary Cutaneous Peripheral T-Cell Lymphoma Not Otherwise Specified: A Rapidly Progressive Variant of Cutaneous T-Cell Lymphoma

    Directory of Open Access Journals (Sweden)

    Kimberly Aderhold

    2015-01-01

    Full Text Available Primary Cutaneous Peripheral T-Cell Lymphoma NOS (PTL-NOS is a rare, progressive, fatal dermatologic disease that presents with features similar to many common benign plaque-like skin conditions, making recognition of its distinguishing features critical for early diagnosis and treatment (Bolognia et al., 2008. A 78-year-old woman presented to ambulatory care with a single 5 cm nodule on her shoulder that had developed rapidly over 1-2 weeks. Examination was suspicious for malignancy and a biopsy was performed. Biopsy results demonstrated CD4 positivity, consistent with Mycosis Fungoides with coexpression of CD5, CD47, and CD7. Within three months her cancer had progressed into diffuse lesions spanning her entire body. As rapid progression is usually uncharacteristic of Mycosis Fungoides, her diagnosis was amended to PTL-NOS. Cutaneous T-Cell Lymphoma (CTCL should be suspected in patients with patches, plaques, erythroderma, or papules that persist or multiply despite conservative treatment. Singular biopsies are often nondiagnostic, requiring a high degree of suspicion if there is deviation from the anticipated clinical course. Multiple biopsies are often necessary to make the diagnosis. Physicians caring for patients with rapidly progressive, nonspecific dermatoses with features described above should keep more uncommon forms of CTCL in mind and refer for early biopsy.

  15. Monitoring and rapid quantification of total carotenoids in Rhodotorula glutinis cells using laser tweezers Raman spectroscopy.

    Science.gov (United States)

    Tao, Zhanhua; Wang, Guiwen; Xu, Xiaodong; Yuan, Yufeng; Wang, Xue; Li, Yongqing

    2011-01-01

    Rhodotorula glutinis is known to accumulate large amounts of carotenoids under certain culture conditions, which have very important industrial applications. So far, the molecular mechanism of regulating carotenogenesis is still not well understood. To better understand the carotenogenesis process, it requires methods that can detect carotenogenesis rapidly and reliably in single live cells. In this paper, a method based on laser tweezers Raman spectroscopy (LTRS) was developed to directly detect carotenoids, as well as other important biological molecules in single live R. glutinis cells. The data showed that the accumulation of carotenoids and lipids occurred mainly in the late exponential and stationary phases when the cell growth was inhibited by nutrient limitation. Meanwhile, the carotenoid concentration changed together with the concentration of nucleic acids, which increased in the first phase and decreased in the last phase of the culture. These data demonstrate that LTRS is a rapid, convenient, and reliable method to study the carotenogenesis process in vivo. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Gene-modified stem cells combined with rapid prototyping techniques: a novel strategy for periodontal regeneration.

    Science.gov (United States)

    He, Huixia; Cao, Junkai; Wang, Dongsheng; Gu, Bing; Guo, Hong; Liu, Hongchen

    2010-03-01

    Periodontal disease, a worldwide prevalent chronic disease in adults, is characterized by the destruction of the periodontal supporting tissue including the cementum, periodontal ligament and alveolar bone. The regeneration of damaged periodontal tissue is the main goal of periodontal treatment. Because conventional periodontal treatments remain insufficient to attain complete and reliable periodontal regeneration, periodontal tissue engineering has emerged as a prospective alternative method for improving the regenerative capacity of periodontal tissue. However, the potential of periodontal regeneration seems to be limited by the understanding of the cellular and molecular events in the formation of periodontal tissue and by the insufficient collaboration of multi-disciplinary research that periodontal tissue engineering involves. In this paper, we first reviewed the recent advancements in stem cells, signaling factors, and scaffolds that relate to periodontal regeneration. Then we speculate that specific genes would improve regenerative capacity of these stem cells, which could differentiate into cementoblasts, osteoblasts and fibroblasts. In addition, the 3D scaffolds that mimic the different structure and physiologic functions of natural fibro-osseous tissue could be fabricated by rapid prototyping (RP) techniques. It was therefore hypothesized that gene-modified stem cells combined with rapid prototyping techniques would be a new strategy to promote more effective and efficient periodontal regeneration.

  17. Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells.

    Science.gov (United States)

    Li, Shengqiao; Yan, Yehong; Lin, Yuan; Bullens, Dominique M; Rutgeerts, Omer; Goebels, Jozef; Segers, Constant; Boon, Louis; Kasran, Ahmad; De Vos, Rita; Dewolf-Peeters, Christiane; Waer, Mark; Billiau, An D

    2007-12-01

    Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.

  18. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

    Science.gov (United States)

    Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

    2017-05-01

    Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10-10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

  19. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

    Science.gov (United States)

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

    2017-08-01

    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  20. Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues.

    Science.gov (United States)

    Wang, Yu; Woehrstein, Johannes B; Donoghue, Noah; Dai, Mingjie; Avendaño, Maier S; Schackmann, Ron C J; Zoeller, Jason J; Wang, Shan Shan H; Tillberg, Paul W; Park, Demian; Lapan, Sylvain W; Boyden, Edward S; Brugge, Joan S; Kaeser, Pascal S; Church, George M; Agasti, Sarit S; Jungmann, Ralf; Yin, Peng

    2017-10-11

    To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

  1. Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

    Science.gov (United States)

    Schmelcher, Mathias; Loessner, Martin J

    2014-01-01

    Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

  2. Transferrin-mediated rapid targeting, isolation, and detection of circulating tumor cells by multifunctional magneto-dendritic nanosystem.

    Science.gov (United States)

    Banerjee, Shashwat S; Jalota-Badhwar, Archana; Satavalekar, Sneha D; Bhansali, Sujit G; Aher, Naval D; Mascarenhas, Russel R; Paul, Debjani; Sharma, Somesh; Khandare, Jayant J

    2013-06-01

    A multicomponent magneto-dendritic nanosystem (MDNS) is designed for rapid tumor cell targeting, isolation, and high-resolution imaging by a facile bioconjugation approach. The highly efficient and rapid-acting MDNS provides a convenient platform for simultaneous isolation and high-resolution imaging of tumor cells, potentially leading towards an early diagnosis of cancer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  4. A simple and rapid flow cytometric method for detection of porcine cell surface markers.

    Science.gov (United States)

    Stabel, T J; Bolin, S R; Pesch, B A; Rahner, T E

    2000-11-01

    The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.

  5. Acute Viral Respiratory Infection Rapidly Induces a CD8+ T Cell Exhaustion-like Phenotype.

    Science.gov (United States)

    Erickson, John J; Lu, Pengcheng; Wen, Sherry; Hastings, Andrew K; Gilchuk, Pavlo; Joyce, Sebastian; Shyr, Yu; Williams, John V

    2015-11-01

    Acute viral infections typically generate functional effector CD8(+) T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus-infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag-induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

    Science.gov (United States)

    Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean

    2017-01-01

    Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

  7. Peripheral blood mononuclear cell gene expression in healthy adults rapidly transported to high altitude

    Directory of Open Access Journals (Sweden)

    Herman NM

    2014-12-01

    Full Text Available Nicole M Herman,1 Diane E Grill,2 Paul J Anderson,1 Andrew D Miller,1 Jacob B Johnson,1 Kathy A O’Malley,1 Maile L Ceridon Richert,1 Bruce D Johnson1 1Department of Cardiovascular Diseases, 2Department of Biostatistics, Mayo Clinic Rochester, MN, USA Abstract: Although mechanisms of high altitude illness have been studied extensively, the processes behind the development of these conditions are still unclear. Few genome-wide studies on rapid exposure to high altitude have been performed. Each year, scientists and support workers are transferred by plane from McMurdo Station in Antarctica (sea level to the Amundsen-Scott South Pole Station at 2,835 meters. This uniform and rapid transfer to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression that may help illustrate the body's adaptations to these conditions. We hypothesized that an extensive number of genes would change with rapid exposure to altitude and further expected that these genes would correspond to inflammatory pathways proposed as a mechanism in development of acute mountain sickness. Peripheral venous blood samples were drawn from 98 healthy subjects at sea level and again on day two at altitude. Microarray analysis was performed on these samples. In total, 1,118 probe sets with significant P-values and fold changes (90% upregulated were identified and entered into MetaCore™ software. Several pathways, including oxidative phosphorylation, cytoskeleton remodeling, and platelet aggregation, were significantly represented by the data set and all were upregulated. Many genes changed expression, and the vast majority of these increased. Increased metabolism in peripheral blood mononuclear cells suggests increased inflammatory activity. Keywords: peripheral blood mononuclear cells, microarray, gene expression, acute mountain sickness

  8. Innate lymphoid cells: models of plasticity for immune homeostasis and rapid responsiveness in protection.

    Science.gov (United States)

    Almeida, F F; Belz, G T

    2016-09-01

    Innate lymphoid cells (ILCs) have stormed onto the immune landscape as "newly discovered" cell types. These tissue-resident sentinels are enriched at mucosal surfaces and engage in complex cross talk with elements of the adaptive immune system and microenvironment to orchestrate immune homeostasis. Many parallels exist between innate cells and T cells leading to the initial partitioning of ILCs into rather rigid subsets that reflect their "adaptive-like" effector cytokines profiles. ILCs themselves, however, have unique attributes that are only just beginning to be elucidated. These features result in complementarity with, rather than complete duplication of, functions of the adaptive immune system. Key transcription factors determine the pathway of differentiation of progenitors towards an ILC1, ILC2, or ILC3 subset. Once formed, flexibility in the responses of these subsets to stimuli unexpectedly allows transdifferentation between the different subsets and the acquisition of altered phenotypes and function. This provides a mechanism for rapid innate immune responsiveness. Here, we discuss the models of differentiation for maintenance and activation of tissue-resident ILCs in maintaining immune homeostasis and protection.

  9. A microfluidic chip for direct and rapid trapping of white blood cells from whole blood

    Science.gov (United States)

    Chen, Jingdong; Chen, Di; Yuan, Tao; Xie, Yao; Chen, Xiang

    2013-01-01

    Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 103 from 3.3 × 103 WBCs. PMID:24404026

  10. Crossroads - Bridging the Digital Divide

    Directory of Open Access Journals (Sweden)

    David Cobb

    2005-03-01

    Full Text Available It’s so good to be in Cambridge, it feels almost like home. Let me start by stating that in the past year ITMB, a successful map publisher in British Columbia, Canada published more paper map titles than at any time in their history. Similarly, the U.S. Geological Survey (USGS recently announced that they have ceased producing paper from their aerial photography archive and will only produce digital copies. I believe that both of these facts speak to the future of maps and digital data. It means there will be paper maps well into the future and there will be an increasing array of digital data - some of it reformatted, as in the USGS case, and most of it will be born digital. When asked to speak about GIS and its role in libraries I often find myself in a conundrum - am I here to slay the dragon, or to pet the dragon. The role of technology in libraries is not one that has been embraced by everyone, and often the technology itself seems to have been force-fed upon us. The library profession is not one that has historically been a proponent of change and the very nature of GIS is change. In one sense, we have been given the choice of becoming paper museums or, at the very least, making GIS technology available in our collections. Today, I would like to review the many ways that GIS is, or will, affect our collections. I will divide the presentation into a general overview of GIS in libraries, how it affects our acquisitions or collection development policies, its effect on cataloging, on reference services, staffing, and our web services. Then I will shift the focus a little and discuss the current situation at the Harvard Map Collection, the future role of legacy collections, and a look to the future.

  11. Active and energy-dependent rapid formation of cell aggregates in the thermophilic photosynthetic bacterium Chloroflexus aggregans.

    Science.gov (United States)

    Hanada, Satoshi; Shimada, Keizo; Matsuura, Katsumi

    2002-03-05

    The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly. The aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20-30 min after the cells were dispersed by shaking. The aggregation depended on the energy supplied by photosynthesis or respiration. Cells aggregated most rapidly under temperature and pH conditions that support maximum growth. The aggregation was also accelerated by the addition of 3-isobutyl-1-methylxanthine that inhibits cyclic 3',5'-AMP phosphodiesterase. Microscopic observation revealed that the bacterium has a fast gliding mobility (1-3 microm s(-1)). The distinctive cell aggregation of C. aggregans was due to this rapid gliding movement.

  12. The Warburg effect as an adaptation of cancer cells to rapid fluctuations in energy demand.

    Directory of Open Access Journals (Sweden)

    Tamir Epstein

    Full Text Available To maintain optimal fitness, a cell must balance the risk of inadequate energy reserve for response to a potentially fatal perturbation against the long-term cost of maintaining high concentrations of ATP to meet occasional spikes in demand. Here we apply a game theoretic approach to address the dynamics of energy production and expenditure in eukaryotic cells. Conventionally, glucose metabolism is viewed as a function of oxygen concentrations in which the more efficient oxidation of glucose to CO2 and H2O produces all or nearly all ATP except under hypoxic conditions when less efficient (2 ATP/ glucose vs. about 36ATP/glucose anaerobic metabolism of glucose to lactic acid provides an emergency backup. We propose an alternative in which energy production is governed by the complex temporal and spatial dynamics of intracellular ATP demand. In the short term, a cell must provide energy for constant baseline needs but also maintain capacity to rapidly respond to fluxes in demand particularly due to external perturbations on the cell membrane. Similarly, longer-term dynamics require a trade-off between the cost of maintaining high metabolic capacity to meet uncommon spikes in demand versus the risk of unsuccessfully responding to threats or opportunities. Here we develop a model and computationally explore the cell's optimal mix of glycolytic and oxidative capacity. We find the Warburg effect, high glycolytic metabolism even under normoxic conditions, is represents a metabolic strategy that allow cancer cells to optimally meet energy demands posed by stochastic or fluctuating tumor environments.

  13. Selective Rapid Eye Movement Sleep Deprivation Affects Cell Size and Number in Kitten Locus Coeruleus

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    James P Shaffery

    2012-05-01

    Full Text Available Cells in the locus coeruleus (LC constitute the sole source of norepinephrine (NE in the brain, and change their discharge rates according to vigilance state. In addition to its well established role in vigilance, NE affects synaptic plasticity in the postnatal critical period (CP of development. One form of CP synaptic plasticity affected by NE results from monocular occlusion, which leads to physiological and cytoarchitectural alterations in central visual areas. Selective suppression of rapid eye movement sleep (REMS in the CP kitten enhances the central effects of monocular occlusion. The mechanisms responsible for heightened cortical plasticity following REMS deprivation (REMSD remain undetermined. One possible mediator of an increase in plasticity is continuous NE outflow, which presumably persists during extended periods of REMSD. Tyrosine hydroxylase (TH is the rate-limiting enzyme in the synthesis of NE and serves as a marker for NE-producing cells. We selectively suppressed REMS in kittens for one week during the CP. The number and size of LC cells expressing immunoreactivity to tyrosine hydroxylase (TH-ir was assessed in age-matched REMS-deprived (RD-, treatment-control (TXC-, and home cage-reared (HCC animals. Sleep amounts and slow wave activity (SWA were also examined relative to baseline. Time spent in REMS during the study was lower in RD compared to TXC animals, and RD kittens increased SWA delta power in the latter half of the REMSD period. The estimated total number of TH-ir cells in LC was significantly lower in the RD- than in the TXC kittens and numerically lower than in HCC animals. The size of LC cells expressing TH-ir was greatest in the HCC group. They were significantly larger than the cells in the RD kittens. These data are consistent with a possible reduction in NE in forebrain areas, including visual cortex, caused by one week of REMSD.

  14. Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood

    Science.gov (United States)

    Sonnenberg, Avery; Marciniak, Jennifer Y.; Rassenti, Laura; Ghia, Emanuela M.; Skowronski, Elaine A.; Manouchehri, Sareh; McCanna, James; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.

    2014-01-01

    BACKGROUND Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a “liquid biopsy” may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification,PCR,and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15–20 mL blood. CONCLUSIONS Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring. PMID:24270796

  15. Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood.

    Science.gov (United States)

    Sonnenberg, Avery; Marciniak, Jennifer Y; Rassenti, Laura; Ghia, Emanuela M; Skowronski, Elaine A; Manouchehri, Sareh; McCanna, James; Widhopf, George F; Kipps, Thomas J; Heller, Michael J

    2014-03-01

    Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required B-cell clone, and then sequenced. PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

  16. Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level.

    Science.gov (United States)

    Harz, M; Rösch, P; Popp, J

    2009-02-01

    Rapid microbial detection and identification with a high grade of sensitivity and selectivity is a great and challenging issue in many fields, primarily in clinical diagnosis, pharmaceutical, or food processing technology. The tedious and time-consuming processes of current microbiological approaches call for faster ideally on-line identification techniques. The vibrational spectroscopic techniques IR absorption and Raman spectroscopy are noninvasive methods yielding molecular fingerprint information; thus, allowing for a fast and reliable analysis of complex biological systems such as bacterial or yeast cells. In this short review, we discuss recent vibrational spectroscopic advances in microbial identification of yeast and bacterial cells for bulk environment and single-cell analysis. IR absorption spectroscopy enables a bulk analysis whereas micro-Raman-spectroscopy with excitation in the near infrared or visible range has the potential for the analysis of single bacterial and yeast cells. The inherently weak Raman signal can be increased up to several orders of magnitude by applying Raman signal enhancement methods such as UV-resonance Raman spectroscopy with excitation in the deep UV region, surface enhanced Raman scattering, or tip-enhanced Raman scattering. Copyright 2008 International Society for Advancement of Cytometry

  17. Rapid Treatment of Leukostasis in Leukemic Mantle Cell Lymphoma Using Therapeutic Leukapheresis: A Case Report

    Directory of Open Access Journals (Sweden)

    Xuan Duc Nguyen

    2011-01-01

    Full Text Available We describe a case of severe leukocytosis caused by leukemic mantle cell lymphoma (MCL, complicated by leukostasis with myocardial infarction in which leukapheresis was used in the initial management. A 73-year-old male presented to the emergency department because of fatigue and thoracic pain. Blood count revealed 630 × 109/L WBC (white blood cells. The electrocardiogram showed ST-elevation with an increase of troponin and creatinine kinase. The diagnosis was ST-elevation myocardial infarction (STEMI induced and complicated by leukostasis. Immunophenotyping, morphology, cytogenetic and fluorescence-in-situ-hybridization analysis revealed the diagnosis of a blastoid variant of MCL. To remove leukocytes rapidly, leukapheresis was performed in the intensive care unit. Based on the differential blood count with 95% blasts, which were assigned to the lymphocyte population by the automatic hematology analyzer, leukapheresis procedures were then performed with the mononuclear cell standard program on the Spectra cell separator. The patient was treated with daily leukapheresis for 3 days. The WBC count decreased to 174 × 109/L after the third leukapheresis, with a 72% reduction. After the second apheresis, treatment with vincristine, cyclophosphamide, and prednisolone was started. The patient fully recovered in the further course of the treatment. To the best of our knowledge, this is the first report on blastoid MCL with leukostasis associated with a STEMI that was successfully treated by leukapheresis. Effective harvest of circulating lymphoma cells by leukapheresis requires adaptation of instrument settings based on the results of the differential blood count prior to apheresis.

  18. Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of Mycobacterium cells in sputum.

    Directory of Open Access Journals (Sweden)

    Shinnosuke Inoue

    Full Text Available An occupationally safe (biosafe sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

  19. Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells.

    Directory of Open Access Journals (Sweden)

    Heather C Jensen-Smith

    Full Text Available Aminoglycosides (AG, including gentamicin (GM, are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs preferentially succumb to multiple HL pathologies while inner hair cells (IHCs are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH fluorescence during acute (1 h GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.

  20. Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling.

    Science.gov (United States)

    Castiello, Luciano; Sabatino, Marianna; Zhao, Yingdong; Tumaini, Barbara; Ren, Jiaqiang; Ping, Jin; Wang, Ena; Wood, Lauren V; Marincola, Francesco M; Puri, Raj K; Stroncek, David F

    2013-02-01

    Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers.

  1. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  2. An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

    Science.gov (United States)

    Doherty, Margaret; Bones, Jonathan; McLoughlin, Niaobh; Telford, Jayne E; Harmon, Bryan; DeFelippis, Michael R; Rudd, Pauline M

    2013-11-01

    Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing

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    Samuel C. Kim

    2015-10-01

    Full Text Available Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 μL samples of a model bacterial strain (E. coli ATCC 25922 treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.

  4. Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

    Science.gov (United States)

    Van Horn, Christopher R; Moretti, Marcelo L; Robertson, Renae R; Segobye, Kabelo; Weller, Stephen C; Young, Bryan G; Johnson, William G; Schulz, Burkhard; Green, Amanda C; Jeffery, Taylor; Lespérance, Mackenzie A; Tardif, François J; Sikkema, Peter H; Hall, J Christopher; McLean, Michael D; Lawton, Mark B; Sammons, R Douglas; Wang, Dafu; Westra, Philip; Gaines, Todd A

    2017-03-07

    Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. Chitosan as coagulant on cyanobacteria in lake restoration management may cause rapid cell lysis.

    Science.gov (United States)

    Mucci, Maíra; Noyma, Natalia Pessoa; de Magalhães, Leonardo; Miranda, Marcela; van Oosterhout, Frank; Guedes, Iamê Alves; Huszar, Vera L M; Marinho, Marcelo Manzi; Lürling, Miquel

    2017-07-01

    Combining coagulant and ballast to remove cyanobacteria from the water column is a promising restoration technique to mitigate cyanobacterial nuisance in surface waters. The organic, biodegradable polymer chitosan has been promoted as a coagulant and is viewed as non-toxic. In this study, we show that chitosan may rapidly compromise membrane integrity and kill certain cyanobacteria leading to release of cell contents in the water. A strain of Cylindrospermopsis raciborskii and one strain of Planktothrix agardhii were most sensitive. A 1.3 h exposure to a low dose of 0.5 mg l-1 chitosan already almost completely killed these cultures resulting in release of cell contents. After 24 h, reductions in PSII efficiencies of all cyanobacteria tested were observed. EC50 values varied from around 0.5 mg l-1 chitosan for the two sensitive strains, via about 5 mg l-1 chitosan for an Aphanizomenon flos-aquae strain, a toxic P. agardhii strain and two Anabaena cylindrica cultures, to more than 8 mg l-1 chitosan for a Microcystis aeruginosa strain and another A. flos-aquae strain. Differences in sensitivity to chitosan might be related to polymeric substances that surround cyanobacteria. Rapid lysis of toxic strains is likely and when chitosan flocking and sinking of cyanobacteria is considered in lake restoration, flocculation efficacy studies should be complemented with investigation on the effects of chitosan on the cyanobacteria assemblage being targeted. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Rapid activation of Rac GTPase in living cells by force is independent of Src.

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    Yeh-Chuin Poh

    2009-11-01

    Full Text Available It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor and YPet (a variant yellow fluorescent protein as an acceptor of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF induced Rac activation at a much later time (>30 sec. There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17 was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src

  7. Rapid labeling of intracellular His-tagged proteins in living cells

    Science.gov (United States)

    Lai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A.; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, Hongzhe

    2015-01-01

    Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells. PMID:25713372

  8. Rapid adhesion of nerve cells to muscle fibers from adult rats is mediated by a sialic acid-binding receptor

    OpenAIRE

    1986-01-01

    Single viable muscle fibers isolated from adult rats by collagenase digestion rapidly bind dissociated spinal neurons or PC-12 cells but not a variety of other cells tested. The adhesion process is calcium- independent, temperature-sensitive, and is not blocked by pretreating cells with inhibitors of energy metabolism or actin polymerization. Adhesion is mediated by a carbohydrate-binding protein and can be inhibited by N-acetylneuraminic acid or mucin, a glycoprotein with high sialic acids c...

  9. A rapid selection strategy for an anodophilic consortium for microbial fuel cells

    KAUST Repository

    Wang, Aijie

    2010-07-01

    A rapid selection method was developed to enrich for a stable and efficient anodophilic consortium (AC) for microbial fuel cells (MFCs). A biofilm sample from a microbial electrolysis cell was serially diluted up to 10-9 in anaerobic phosphate buffer solution and incubated in an Fe(III)-acetate medium, and an Fe(III)-reducing AC was obtained for dilutions up to 10-6. The activity of MFC inoculated with the enrichment AC was compared with those inoculated with original biofilm or activated sludge. The power densities and Coulombic efficiencies of the AC (226 mW/m2, 34%) were higher than those of the original biofilm (209 mW/m2, 23%) and activated sludge (192 mW/m2, 19%). The start-up period of the AC (60 h) was also shorter than those obtained with the other inocula (biofilm, 95 h; activated sludge, 300 h). This indicated that such a strategy is highly efficient for obtaining an anodophilic consortium for improving the performance of an MFC. © 2010 Elsevier Ltd.

  10. Cell-free expression of protein kinase a for rapid activity assays.

    Science.gov (United States)

    Leippe, Donna M; Zhao, Kate Qin; Hsiao, Kevin; Slater, Michael R

    2010-05-19

    Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag((R)) fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

  11. Cell-Free Expression of Protein Kinase a for Rapid Activity Assays

    Directory of Open Access Journals (Sweden)

    Donna M. Leippe

    2010-01-01

    Full Text Available Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag ® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

  12. Cell-Free Expression of Protein Kinase A for Rapid Activity Assays

    Directory of Open Access Journals (Sweden)

    Donna M. Leippe

    2010-05-01

    Full Text Available Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

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

    Directory of Open Access Journals (Sweden)

    Yusuke Sakai

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

  14. Quantitative Measurement of the Digital Divide

    Science.gov (United States)

    Cottrell, Roger

    2007-04-01

    Bandwidth and the Internet infrastructure are the life-blood of the world's knowledge economy, but they are often scarcest where most needed. Measuring the numbers of users of the Internet infrastructure is not easy in developing countries because many people share accounts, use corporate and academic networks, or visit the rapidly growing number of cyber cafes, telecentres and business services. Also measuring the number of users does not take into account the level of use. One valuable indicator for measuring the Internet infrastructure is the international Internet performance of a country or region. One of the major aims of the PingER project is to provide an historical archive of extensive, publicly accessible, up-to-date, measurements, analyses and reports of multiple Internet performance indicators (such as delay, loss, throughput, reachability, and jitter) between sites, countries and regions of the world. This talk will briefly describe the PingER project and then compare and contrast the Internet performance and its trends within and between countries and regions of the world. By means of extensive case studies it will also identify which regions need the greatest attention, together with their major issues and possible approaches to reducing the divide.

  15. Rapid auxin-induced stimulation of cell wall synthesis in pea internodes

    Energy Technology Data Exchange (ETDEWEB)

    Kutschera, U.; Briggs, W.R.

    1987-05-01

    The effect of auxin (indole-3-acetic acid; IAA) on growth and incorporation of myo-(2-/sup 3/H(N)) inositol ((/sup 3/H)Ins) into noncellulosic polysacchharides in the cell walls of third internode sections from red light-grown pea seedlings (Pisum sativum L. cv. Alaska) was investigated. Intact section were incubated on (/sup 3/H)Ins for 4 hr to permit uptake of the tracer and then IAA was added. Growth started after a lag phase of 15 min under these conditions. The sections were removed from the tracer and separated into epidermis and cortical cylinder (cortex plus vascular tissue). In the epidermis, IAA-induced stimulation of (/sup 3/H)Ins incorporation started after a lag of 15 min. The amount of incorporation was 15% higher after 30 min and 24% higher after 2 hr than in the control. In the cortical cylinder, IAA-induced stimulation of (/sup 3/H)Ins incorporation started only approx. = 1 hr after adding IAA. The ionophore monensin (20 ..mu..M) inhibited the IAA-induced growth by 95%. Under these conditions, the IAA-induced stimulation of (/sup 3/H)Ins incorporation and the IAA-induced increase in in vivo extensibility of the sections was almost completely inhibited, although oxygen uptake was unaffected. The authors suggest that wall synthesis (as represented by (/sup 3/H)Ins incorporation) and wall loosening (increase in in vivo extensibility) are related processes. The results support the hypothesis that IAA induces growth by rapid simulation of cell wall synthesis in the growth-limiting epidermal cell layer.

  16. A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model.

    Science.gov (United States)

    Tu, Shih-Hsin; Hsieh, Yi-Chen; Huang, Li-Chi; Lin, Chun-Yu; Hsu, Kai-Wen; Hsieh, Wen-Shyang; Chi, Wei-Ming; Lee, Chia-Hwa

    2017-06-23

    As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models. In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D-imaging on orthotropic-developed breast-tumor-bearing mice. In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x10(11) cell numbers with great correlation (R(2) = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R(2) > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0

  17. Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

    Science.gov (United States)

    Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien

    2015-04-01

    The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo

    Science.gov (United States)

    Vukmanovic-Stejic, Milica; Zhang, Yan; Cook, Joanne E.; Fletcher, Jean M.; McQuaid, Arthur; Masters, Joanne E.; Rustin, Malcolm H.A.; Taams, Leonie S.; Beverley, Peter C.L.; Macallan, Derek C.; Akbar, Arne N.

    2006-01-01

    While memory T cells are maintained by continuous turnover, it is not clear how human regulatory CD4+CD45RO+CD25hi Foxp3+ T lymphocyte populations persist throughout life. We therefore used deuterium labeling of cycling cells in vivo to determine whether these cells could be replenished by proliferation. We found that CD4+CD45RO+Foxp3+CD25hi T lymphocytes were highly proliferative, with a doubling time of 8 days, compared with memory CD4+CD45RO+Foxp3–CD25– (24 days) or naive CD4+CD45RA+Foxp3–CD25– populations (199 days). However, the regulatory population was susceptible to apoptosis and had critically short telomeres and low telomerase activity. It was therefore unlikely to be self regenerating. These data are consistent with continuous production from another population source. We found extremely close TCR clonal homology between regulatory and memory CD4+ T cells. Furthermore, antigen-related expansions within certain TCR Vβ families were associated with parallel numerical increases of CD4+CD45RO+CD25hiFoxp3+ Tregs with the same Vβ usage. It is therefore unlikely that all human CD4+CD25+Foxp3+ Tregs are generated as a separate functional lineage in the thymus. Instead, our data suggest that a proportion of this regulatory population is generated from rapidly dividing, highly differentiated memory CD4+ T cells; this has considerable implications for the therapeutic manipulation of these cells in vivo. PMID:16955142

  19. The road to sustainability must bridge three great divides.

    Science.gov (United States)

    Aronson, James; Blignaut, James N; de Groot, Rudolf S; Clewell, Andre; Lowry, Porter P; Woodworth, Paddy; Cowling, Richard M; Renison, Daniel; Farley, Joshua; Fontaine, Christelle; Tongway, David; Levy, Samuel; Milton, Suzanne J; Rangel, Orlando; Debrincat, Bev; Birkinshaw, Chris

    2010-01-01

    The world's large and rapidly growing human population is exhausting Earth's natural capital at ever-faster rates, and yet appears mostly oblivious to the fact that these resources are limited. This is dangerous for our well-being and perhaps for our survival, as documented by numerous studies over many years. Why are we not moving instead toward sustainable levels of use? We argue here that this disconnection between our knowledge and our actions is largely caused by three "great divides": an ideological divide between economists and ecologists; an economic development divide between the rich and the poor; and an information divide, which obstructs communications between scientists, public opinion, and policy makers. These divides prevent our economies from responding effectively to urgent signals of environmental and ecological stress. The restoration of natural capital (RNC) can be an important strategy in bridging all of these divides. RNC projects and programs make explicit the multiple and mutually reinforcing linkages between environmental and economic well-being, while opening up a promising policy road in the search for a sustainable and desirable future for global society. The bridge-building capacity of RNC derives from its double focus: on the ecological restoration of degraded, overexploited natural ecosystems, and on the full socio-economic and ecological interface between people and their environments.

  20. Reactive oxygen species regulatory mechanisms associated with rapid response of MC3T3-E1 cells for vibration stress

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ling; Gan, Xueqi; Zhu, Zhuoli; Yang, Yang; He, Yuting; Yu, Haiyang, E-mail: yhyang6812@scu.edu.cn

    2016-02-12

    Although many previous studies have shown that refractory period-dependent memory effect of vibration stress is anabolic for skeletal homeostasis, little is known about the rapid response of osteoblasts simply derived from vibration itself. In view of the potential role of reactive oxygen species (ROS) in mediating differentiated activity of osteoblasts, whether and how ROS regulates the rapid effect of vibration deserve to be demonstrated. Our findings indicated that MC3T3-E1 cells underwent decreased gene expression of Runx2, Col-I and ALP and impaired ALP activity accompanied by increased mitochondrial fission immediately after vibration loading. Moreover, we also revealed the involvement of ERK-Drp1 signal transduction in ROS regulatory mechanisms responsible for the rapid effect of vibration stress. - Highlights: • ROS contributed to the rapid response of MC3T3-E1 cells for vibration stress. • Imbalance of mitochondrial dynamics were linked to the LMHFV-derived rapid response. • The role of ERK-Drp1 signal pathway in the LMHFV-derived osteoblast rapid response.

  1. Global patent landscape of programmed cell death 1: implications of the rapid expansion.

    Science.gov (United States)

    Kong, Xiangjun; Zhang, Qianru; Lai, Yunfeng; Hu, Hao; Chen, Xin; Hu, Yuanjia

    2018-01-01

    Inhibitors of programmed cell death 1 (PD-1) and its ligands are producing a paradigm shift in cancer treatment. The promising clinical outcomes and a multi-billion dollar market have prompted active research and development and resulted in relentless patent protection. However, the global patent landscape in this field remains unclear. Areas covered: The patent landscape encompassing global patenting activities and developing trends in the field is discussed based on a data set of 1287 patent families. Patenting activities have expanded rapidly in the past three years. Specific trends in relevant aspects are presented, including patent filing countries, patent ownership, co-patents, technical areas, and technological connections in terms of patent citation relationships. Expert opinion: Together with patenting momentum in recent years, fragmented ownership and dense technological connections of PD-1-related inventions raise the possibility of a patent thicket. The explosion of patent applications and complex citation relationships could also lead to considerable patent conflicts and disputes on overlapping intellectual property rights, in addition to existing legal uncertainties. Patent applicants in this field are encouraged to be aware of these concerns when developing valid patent strategies.

  2. Rapid identification of mRNA processing defects with a novel single-cell yeast reporter.

    Science.gov (United States)

    Sorenson, Matthew R; Stevens, Scott W

    2014-05-01

    It has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter using multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 nonessential yeast genes revealed striking phenotypic overlaps between chromatin remodeling, histone modification, and pre-mRNA splicing. Additionally, we developed a copper-inducible reporter, with which we demonstrate that 5-fluorouracil mimics the mRNA decay phenotype of cells lacking the 3'-5' exonuclease Rrp6p. Our reporter is capable of performing high-throughput, rapid, and large-scale screens to identify and characterize genetic and chemical perturbations of the major eukaryotic gene expression processes.

  3. Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV

    Science.gov (United States)

    Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki

    2016-01-01

    Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

  4. Rabbit antithymocyte globulin induces rapid expansion of effector memory CD8 T cells without accelerating acute graft versus host disease.

    Science.gov (United States)

    Wittenbecher, Friedrich; Rieger, Kathrin; Dziubianau, Mikalai; Herholz, Anne; Mensen, Angela; Blau, Igor Wolfgang; Uharek, Lutz; Dörken, Bernd; Thiel, Andreas; Na, Il-Kang

    2013-01-01

    Rabbit antithymocyte globulin (Thymoglobulin(®)) is commonly used as graft-versus-host disease (GvHD) prophylaxis. Since we found similar total CD8 T cell numbers in patients with and without Thymoglobulin(®) therapy within the first six months after allogeneic hematopoietic stem cell transplantation, we have analyzed the reconstitution of the CD8 T cell compartment in detail. After T cell-depletion, higher and more sustained proliferative capacity of memory CD8 T cells resulted in their rapid expansion, whereas the fraction of naive CD8 T cells decreased. Importantly, this shift towards effector memory CD8 T cells did not accelerate the incidence of GvHD.

  5. Investigation of Near-Surface Defects Induced by Spike Rapid Thermal Annealing in c-SILICON Solar Cells

    Science.gov (United States)

    Liu, Guodong; Ren, Pan; Zhang, Dayong; Wang, Weiping; Li, Jianfeng

    2016-01-01

    The defects induced by a spike rapid thermal annealing (RTA) process in crystalline silicon (c-Si) solar cells were investigated by the photoluminescence (PL) technique and the transmission electron microscopy (TEM), respectively. Dislocation defects were found to form in the near-surface junction region of the monocrystalline Si solar cell after a spike RTA process was performed at 1100∘C. Photo J-V characteristics were measured on the Si solar cell before and after the spike RTA treatments to reveal the effects of defects on the Si cell performances. In addition, the Silvaco device simulation program was used to study the effects of defects density on the cell performances by fitting the experimental data of RTA-treated cells. The results demonstrate that there was an obvious degradation in the Si solar cell performances when the defect density after the spike RTA treatment was above 1×1013cm-3.

  6. Fast frequency divider circuit using combinational logic

    Science.gov (United States)

    Helinski, Ryan

    2017-05-30

    The various technologies presented herein relate to performing on-chip frequency division of an operating frequency of a ring oscillator (RO). Per the various embodiments herein, a conflict between RO size versus operational frequency can be addressed by dividing the output frequency of the RO to a frequency that can be measured on-chip. A frequency divider circuit (comprising NOR gates and latches, for example) can be utilized in conjunction with the RO on the chip. In an embodiment, the frequency divider circuit can include a pair of latches coupled to the RO to facilitate dividing the oscillating frequency of the RO by 2. In another embodiment, the frequency divider circuit can include four latches (operating in pairs) coupled to the RO to facilitate dividing the oscillating frequency of the RO by 4. A plurality of ROs can be MUXed to the plurality of ROs by a single oscillation-counting circuit.

  7. Development of an OP9 derived cell line as a robust model to rapidly study adipocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Jacqueline M Lane

    Full Text Available One hallmark of obesity is adipocyte hypertrophy and hyperplasia. To gain novel insights into adipose biology and therapeutics, there is a pressing need for a robust, rapid, and informative cell model of adipocyte differentiation for potential RNAi and drug screens. Current models are prohibitive for drug and RNAi screens due to a slow differentiation time course and resistance to transfection. We asked if we could create a rapid, robust model of adipogenesis to potentially enable rapid functional and obesity therapeutic screens. We generated the clonal population OP9-K, which differentiates rapidly and reproducibly, and displays classic adipocyte morphology: rounded cell shape, lipid accumulation, and coalescence of lipids into a large droplet. We further validate the OP9-K cells as an adipocyte model system by microarray analysis of the differentiating transcriptome. OP9-K differentiates via known adipogenic pathways, involving the transcriptional activation and repression of common adipose markers Plin1, Gata2, C/Ebpα and C/Ebpβ and biological pathways, such as lipid metabolism, PPARγ signaling, and osteogenesis. We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ. We further utilized our model to query the effects of a novel obesity therapeutic target, the transcription factor SPI1. We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation. We demonstrate rapid, robust differentiation and efficient transfectability of the OP9-K cell model of adipogenesis. Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time. The results of this study can contribute to the development of rapid screens with the potential to deepen our understanding of adipose biology and efficiently

  8. Digital divide and body size disparities among Chinese adults

    OpenAIRE

    Chih-Chien Huang; Scott T. Yabiku

    2018-01-01

    Background: The rapid development of information and communication technology (ICT) in China has increased people's sedentary behavior and raised a number of related issues. ICT screen-viewing activities are increasingly considered to contribute to obesity, and sociodemographic characteristics such as gender, income, age, education, and geographical location seem to magnify the digital divide. Objective: This study first examines dissimilar stages of ICT transition, and then establishes ho...

  9. Optimization of electrospun poly(N-isopropyl acrylamide) mats for the rapid reversible adhesion of mammalian cells.

    Science.gov (United States)

    Cicotte, Kirsten N; Reed, Jamie A; Nguyen, Phuong Anh H; De Lora, Jacqueline A; Hedberg-Dirk, Elizabeth L; Canavan, Heather E

    2017-06-13

    Poly(N-isopropyl acrylamide) (pNIPAM) is a "smart" polymer that responds to changes in altering temperature near physiologically relevant temperatures, changing its relative hydrophobicity. Mammalian cells attach to pNIPAM at 37 °C and detach spontaneously as a confluent sheet when the temperature is shifted below the lower critical solution temperature (∼32 °C). A variety of methods have been used to create pNIPAM films, including plasma polymerization, self-assembled monolayers, and electron beam ionization. However, detachment of confluent cell sheets from these pNIPAM films can take well over an hour to achieve potentially impacting cellular behavior. In this work, pNIPAM mats were prepared via electrospinning (i.e., espNIPAM) by a previously described technique that the authors optimized for cell attachment and rapid cell detachment. Several electrospinning parameters were varied (needle gauge, collection time, and molecular weight of the polymer) to determine the optimum parameters. The espNIPAM mats were then characterized using Fourier-transform infrared, x-ray photoelectron spectroscopy, and scanning electron microscopy. The espNIPAM mats showing the most promise were seeded with mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once confluent, the temperature of the cells and mats was changed to ∼25 °C, resulting in the extremely rapid swelling of the mats. The authors find that espNIPAM mats fabricated using small, dense fibers made of high molecular weight pNIPAM are extremely well-suited as a rapid release method for cell sheet harvesting.

  10. Rapid hyperfractionated radiotherapy. Clinical results in 178 advanced squamous cell carcinomas of the head and neck

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, T.D.; Demange, L.; Froissart, D.; Panis, X.; Loirette, M.

    1985-07-01

    The authors present a series of 178 patients with Stage III or IV squamous cell carcinoma of the head and neck treated by rapid irradiation using multiple and small fractions per day. An initial group of 91 patients (G1) received a total dose of 72 Gy in 80 sessions and 10 days, according to the following split course schedule: J1 to J5, 36 Gy in 40 sessions, eight daily fractions of .9 Gy separated by 2 hours; J6 to J20, rest period; J21 to J25, same as in J1 except that the spinal cord was shielded. This protocol was altered for the following 87 patients (G2) by lessening the total dose to 60 to 66 Gy and the number of fractions to 60. The rest period was lengthened to 4 weeks. All patients but five completed the whole program and the minimal follow-up period was 24 months. At the end of irradiation, 121 patients achieved a total remission, but local recurrences occurred in 56%. Moreover, acute intolerance was considered as severe in 34% of G1 patients, and included extensive mucosal necrosis and bleeding. Although this rate was significantly reduced in G2 patients, late complications were observed in 20 of the 25 survivors, and included trismus, cervical sclerosis, and recurrent laryngeal edema. The crude survival rate is 13% at 2 years. Although this study was not randomized, this particular type of accelerated and hyperfractionated combination of irradiation did not really improve the clinical results in advanced carcinoma of the head and neck. Other schedules and probably other tumors, less extended, should be tested.

  11. Endothelial Cells Are Susceptible to Rapid siRNA Transfection and Gene Silencing Ex Vivo

    Science.gov (United States)

    Andersen, Nicholas D.; Chopra, Atish; Monahan, Thomas S.; Malek, Junaid Y.; Jain, Monica; Pradhan, Leena; Ferran, Christiane; LoGerfo, Frank W.

    2010-01-01

    BACKGROUND Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room. MATERIALS AND METHODS Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mmHg hyperbaric pressure, or (iii) 120 mmHg luminal distending pressure. Transfected vein segments were maintained in organ culture. siRNA delivery and gene silencing were assessed by tissue layer using confocal microscopy and immunohistochemistry. RESULTS Distending pressure transfection yielded the highest levels of endothelial siRNA delivery (22% pixels fluorescing) and gene silencing (60% GAPDH knockdown, 55% eNOS knockdown) as compared to hyperbaric (12% pixels fluorescing, 36% GAPDH knockdown, 30% eNOS knockdown) or non-pressurized transfections (10% pixels fluorescing, 30% GAPDH knockdown, 25% eNOS knockdown). Cumulative endothelial siRNA delivery (16% pixels fluorescing) and gene silencing (46% GAPDH knockdown) exceeded levels achieved in the media/adventitia (8% pixels fluorescing, 24% GAPDH knockdown) across all transfection methods. CONCLUSION Endothelial gene silencing is possible within the timeframe and conditions of surgical application without the use of transfection reagents. The high sensitivity of endothelial cells to siRNA transfection marks the endothelium as a promising target of gene therapy in vascular disease. PMID:20801607

  12. New Media and the Digital Divide

    NARCIS (Netherlands)

    van Deursen, Alexander Johannes Aloysius Maria; van Dijk, Johannes A.G.M.; Wright, James D.

    2015-01-01

    During the 1990s, researchers and policy makers began discussing the presence of a so-called ‘digital divide,’ a distinction of people who do and do not have access to information and communication technologies. The concept of the digital divide stems from a comparative perspective of social and

  13. Social Welfare Implications of the Digital Divide

    Science.gov (United States)

    Kim, Eunjin; Lee, Byungtae; Menon, Nirup M.

    2009-01-01

    The Internet plays a critical role in informing individuals about society, politics, business, and the environment. So much so that it has been said that the digital divide makes the segment of society on the ''right side'' of the divide (the digitally endowed group) better off and that on the ''wrong side'' (the digitally challenged group) worse…

  14. Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette Tina Marie [Los Alamos National Laboratory

    2008-01-01

    The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

  15. α6 Integrin (α6high/Transferrin Receptor (CD71low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

    Directory of Open Access Journals (Sweden)

    Elodie Metral

    2017-01-01

    Full Text Available The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC and Transitory Amplifying (TA cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i the rapid adhesion method on coated substrate and (ii the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71. Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.

  16. Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo.

    Science.gov (United States)

    Li, Baoqiang; Wang, Lei; Xu, Feng; Gang, Xiaomin; Demirci, Utkan; Wei, Daqing; Li, Ying; Feng, Yujie; Jia, Dechang; Zhou, Yu

    2015-08-01

    Natural and biodegradable chitosan with unique amino groups has found widespread applications in tissue engineering and drug delivery. However, its applications have been limited by the poor solubility of native chitosan in neutral pH solution, which subsequently fails to achieve cell-laden hydrogel at physiological pH. To address this, we incorporated UV crosslinking ability in chitosan, allowing fabrication of patterned cell-laden and rapid transdermal curing hydrogel in vivo. The hydrosoluble, UV crosslinkable and injectable N-methacryloyl chitosan (N-MAC) was synthesized via single-step chemoselective N-acylation reaction, which simultaneously endowed chitosan with well solubility in neutral pH solution, UV crosslinkable ability and injectability. The solubility of N-MAC in neutral pH solution increased 2.21-fold with substitution degree increasing from 10.9% to 28.4%. The N-MAC allowed fabrication of cell-laden microgels with on-demand patterns via photolithography, and the cell viability in N-MAC hydrogel maintained 96.3 ± 1.3% N-MAC allowed rapid transdermal curing hydrogel in vivo within 60s through minimally invasive clinical surgery. Histological analysis revealed that low-dose UV irradiation hardly induced skin injury and acute inflammatory response disappeared after 7 days. N-MAC would allow rapid, robust and cost-effective fabrication of patterned cell-laden polysaccharide microgels with unique amino groups serving as building blocks for tissue engineering and rapid transdermal curing hydrogel in vivo for localized and sustained protein delivery. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  17. Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids.

    Science.gov (United States)

    Liu, Xiaomei; Ping, Han; Zhang, Chun

    2014-09-10

    Stable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 (AAV2) Rep protein is known to induce rAAV DNA integration into a specific site (AAVS1) of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line. We have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII (FVIII-BDD) and GFP gene flanked by AAV ITRs. Human embryonic kidney (HEK) 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity. We developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.

  18. Transcriptome Signatures Reveal Rapid Induction of Immune-Responsive Genes in Human Memory CD8(+) T Cells.

    Science.gov (United States)

    Yang, Cheng; Khanniche, Asma; DiSpirito, Joanna R; Ji, Ping; Wang, Shujun; Wang, Ying; Shen, Hao

    2016-05-31

    Memory T cells (TM) play a prominent role in protection and auto-immunity due to their ability to mount a more effective response than naïve T cells (TN). However, the molecular mechanisms underlying enhanced functionality of TM are not well defined, particularly in human TM. We examined the global gene expression profiles of human CD8(+) TN and TM before and after stimulation. There were 1,284, 1,373 and 1,629 differentially expressed genes between TN and TM at 0 hr, 4 hr and 24 hr after stimulation, respectively, with more genes expressed to higher levels in TM. Genes rapidly up-regulated in TN cells were largely involved in nitrogen, nucleoside and amino acid metabolisms. In contrast, those in CD8(+) TM were significantly enriched for immune-response-associated processes, including cytokine production, lymphocyte activation and chemotaxis. Multiple cytokines were rapidly up-regulated in TM cells, including effector cytokines known to be produced by CD8(+) T cells and important for their functions, as well as regulatory cytokines, both pro- and anti-inflammatory, that are not typically produced by CD8(+) T cells. These results provide new insights into molecular mechanisms that contribute to the enhanced functionality of human CD8(+) TM and their prominent role in protection and auto-immunity.

  19. Rapid fabrication of microfluidic polymer electrolyte membrane fuel cell in PDMS by surface patterning of perfluorinated ion-exchange resin

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yong-Ak; Han, Jongyoon [Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States); Batista, Candy [Roxbury Community College, 1234 Columbus Ave., Roxbury Crossing, MA 02120 (United States); Sarpeshkar, Rahul [Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States)

    2008-09-01

    In this paper we demonstrate a simple and rapid fabrication method for a microfluidic polymer electrolyte membrane (PEM) fuel cell using polydimethylsiloxane (PDMS), which has become the de facto standard material in BioMEMS. Instead of integrating a Nafion sheet film between two layers of a PDMS device in a traditional ''sandwich format,'' we pattern a perfluorinated ion-exchange resin such as a Nafion resin on a glass substrate using a reversibly bonded PDMS microchannel to generate an ion-selective membrane between the fuel-cell electrodes. After this patterning step, the assembly of the microfluidic fuel cell is accomplished by simple oxygen plasma bonding between the PDMS chip and the glass substrate. In an example implementation, the planar PEM microfluidic fuel cell generates an open circuit voltage of 600-800 mV and delivers a maximum current output of nearly 4 {mu}A. To enhance the power output of the fuel cell we utilize self-assembled colloidal arrays as a support matrix for the Nafion resin. Such arrays allow us to increase the thickness of the ion-selective membrane to 20 {mu}m and increase the current output by 166%. Our novel fabrication method enables rapid prototyping of microfluidic fuel cells to study various ion-exchange resins for the polymer electrolyte membrane. Our work will facilitate the development of miniature, implantable, on-chip power sources for biomedical applications. (author)

  20. Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge.

    Science.gov (United States)

    Krishnamurty, Akshay T; Thouvenel, Christopher D; Portugal, Silvia; Keitany, Gladys J; Kim, Karen S; Holder, Anthony; Crompton, Peter D; Rawlings, David J; Pepper, Marion

    2016-08-16

    Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol.

    Science.gov (United States)

    Chen, Yu-Fan; Tseng, Chien-Yu; Wang, Hsei-Wei; Kuo, Hung-Chih; Yang, Vincent W; Lee, Oscar K

    2012-04-01

    Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases. Copyright © 2011 American Association for the Study of Liver Diseases.

  2. Digital divide in Serbia from demographic perspective

    Directory of Open Access Journals (Sweden)

    Milovanović Slavoljub M.

    2013-01-01

    Full Text Available Digital divide is a term that defines differences and inequality between various groups of population considering opportunities and abilities for access and use of information and communication technologies (ICT, particularly the Internet. Digital divide can be viewed from regional and global aspect, so there are significant differences between regions in a country and between countries, considering the use of ICT. On the global level, there are the countries where a great part of the population has access to ICT and countries where that is not the case. Serbia as a country that is in transition period belongs to the second group of countries. In addition, digital divide exists on a country level, because in south-east region of Serbia relatively small number of people has access to ICT comparing to the other regions. The paper just treats problem of digital divide on the example of Republic of Serbia, with specific view to demographic aspect of the problem.

  3. Environmental Aesthetics. Crossing Divides and Breaking Ground

    NARCIS (Netherlands)

    Drenthen, M.; Keulartz, J.

    2014-01-01

    Environmental aesthetics crosses several commonly recognized divides: between analytic and continental philosophy, Eastern and Western traditions, universalizing and historicizing approaches, and theoretical and practical concerns. This volume sets out to show how these,perspectives can be brought

  4. All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

    KAUST Repository

    So, Hongyun

    2014-11-24

    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

  5. Rapid Antibiotic Susceptibility Testing of Uropathogenic E. coli by Tracking Submicron Scale Motion of Single Bacterial Cells.

    Science.gov (United States)

    Syal, Karan; Shen, Simon; Yang, Yunze; Wang, Shaopeng; Haydel, Shelley E; Tao, Nongjian

    2017-08-25

    To combat antibiotic resistance, a rapid antibiotic susceptibility testing (AST) technology that can identify resistant infections at disease onset is required. Current clinical AST technologies take 1-3 days, which is often too slow for accurate treatment. Here we demonstrate a rapid AST method by tracking sub-μm scale bacterial motion with an optical imaging and tracking technique. We apply the method to clinically relevant bacterial pathogens, Escherichia coli O157: H7 and uropathogenic E. coli (UPEC) loosely tethered to a glass surface. By analyzing dose-dependent sub-μm motion changes in a population of bacterial cells, we obtain the minimum bactericidal concentration within 2 h using human urine samples spiked with UPEC. We validate the AST method using the standard culture-based AST methods. In addition to population studies, the method allows single cell analysis, which can identify subpopulations of resistance strains within a sample.

  6. Divide and Recombine for Large Complex Data

    Science.gov (United States)

    2017-12-01

    SUPPLEMENTARY NOTES 14. ABSTRACT Divide and Recombine (D& R ) statistical approach was developed for analyzing ‘big data’ where the computational complexity...is very high. The analyst divides data into subsets by a D& R division technique, applying analytic methods to each subset independently, without...communication. Outputs of each analytic method are recombined by a D& R recombination procedure, which allows extensive parallel computation. DeltaRho

  7. Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2008-11-01

    The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca(2+) absorptive pathways involved in the regulation of whole body Ca(2+) homeostasis. The role of steroid hormone 17beta-estradiol (E(2)), in [Ca(2+)](i) regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologous systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E(2) in [Ca(2+)](i) regulation involving directly TRPV6 channels in T84 cells. Treatment with E(2) rapidly (<5 min) enhanced [Ca(2+)](i) and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E(2), Ca(2+) enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca(2+) entry\\/signalling pathway that is sensitive to 17beta-estradiol.

  8. Rapid prototyping of nano- and micro-patterned substrates for the control of cell neuritogenesis by topographic and chemical cues

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Ajay V.; Gailite, Lasma; Vyas, Varun [European School of Molecular Medicine (SEMM), IFOM-IEO Campus, Via Adamello 16, I-20139 Milano (Italy); CIMAINA and Dipartimento di Fisica, Universita di Milano, via Celoria 16, I-20133 Milano (Italy); Lenardi, Cristina, E-mail: cristina.lenardi@mi.infn.it [CIMAINA and Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Universita di Milano, via Trentacoste 2, I-20134 Milano (Italy); Fondazione Filarete, viale Ortles 22/4, I-20139 Milano (Italy); Forti, Stefania [CIMAINA and Dipartimento di Fisica, Universita di Milano, via Celoria 16, I-20133 Milano (Italy); Matteoli, Michela [Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita di Milano, via Vanvitelli 32, I-20139 Milano (Italy); Fondazione Filarete, viale Ortles 22/4, I-20139 Milano (Italy); Milani, Paolo, E-mail: paolo.milani@mi.infn.it [CIMAINA and Dipartimento di Fisica, Universita di Milano, via Celoria 16, I-20133 Milano (Italy); Fondazione Filarete, viale Ortles 22/4, I-20139 Milano (Italy)

    2011-07-20

    Rapid prototyping of titania substrates with micro and nanofeatures is obtained by combining nanosphere lithography with supersonic cluster beam deposition on protein-functionalized glass supports. The proliferation and differentiation of PC12 cells were studied on these substrates. The facile control and modification of the substrate structure at the micro- and nanoscale allowed us to characterize the role of functional and structural features on neuritogenesis and to control this phenomenon by identifying the optimal topography.

  9. A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix.

    Science.gov (United States)

    Hellewell, Andrew L; Rosini, Silvia; Adams, Josephine C

    2017-01-04

    The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation. Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

  10. Rapid progression of mediastinal tumor within a few days: A case report of T cell lymphoblastic lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Tae Ran; Lee, Young Kyung; Jun, Hyun Jung; Jung, Eun Ah; Son, Jin Sung [Seoul Medical Center, Seoul (Korea, Republic of)

    2016-05-15

    T-cell lymphoblastic lymphoma is a highly aggressive tumor derived from lymphocyte of the thymus, which accounts for 2% of non-Hodgkin's lymphoma. The disease occurs most commonly in adolescent and young adult males. It often results in respiratory emergency because of high proliferation rate. In this case, we confirmed the rapid progression of T-cell lymphoblastic lymphoma through the chest CT scan with one week interval. Three days of empirical chemotherapy resulted in substantial reduction of mediastinal mass, pleural thickening and pleural effusion.

  11. Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology

    Directory of Open Access Journals (Sweden)

    Sven Kruspe

    2017-09-01

    Full Text Available A challenge for circulating tumor cell (CTC-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1 their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2 their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers.

  12. Rapid Conversion from Carbohydrates to Large-Scale Carbon Quantum Dots for All-Weather Solar Cells.

    Science.gov (United States)

    Tang, Qunwei; Zhu, Wanlu; He, Benlin; Yang, Peizhi

    2017-02-28

    A great challenge for state-of-the-art solar cells is to generate electricity in all weather. We present here the rapid conversion of carbon quantum dots (CQDs) from carbohydrates (including glucose, maltol, sucrose) for an all-weather solar cell, which comprises a CQD-sensitized mesoscopic titanium dioxide/long-persistence phosphor (m-TiO 2 /LPP) photoanode, a I - /I 3 - redox electrolyte, and a platinum counter electrode. In virtue of the light storing and luminescent behaviors of LPP phosphors, the generated all-weather solar cells can not only convert sunlight into electricity on sunny days but persistently realize electricity output in all dark-light conditions. The maximized photoelectric conversion efficiency is as high as 15.1% for so-called all-weather CQD solar cells in dark conditions.

  13. Precise spatio-temporal control of rapid optogenetic cell ablation with mem-KillerRed in Zebrafish.

    Science.gov (United States)

    Buckley, C; Carvalho, M T; Young, L K; Rider, S A; McFadden, C; Berlage, C; Verdon, R F; Taylor, J M; Girkin, J M; Mullins, J J

    2017-07-11

    The ability to kill individual or groups of cells in vivo is important for studying cellular processes and their physiological function. Cell-specific genetically encoded photosensitizing proteins, such as KillerRed, permit spatiotemporal optogenetic ablation with low-power laser light. We report dramatically improved resolution and speed of cell targeting in the zebrafish kidney through the use of a selective plane illumination microscope (SPIM). Furthermore, through the novel incorporation of a Bessel beam into the SPIM imaging arm, we were able to improve on targeting speed and precision. The low diffraction of the Bessel beam coupled with the ability to tightly focus it through a high NA lens allowed precise, rapid targeting of subsets of cells at anatomical depth in live, developing zebrafish kidneys. We demonstrate that these specific targeting strategies significantly increase the speed of optoablation as well as fish survival.

  14. Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic β-Cells

    Science.gov (United States)

    Nakagawa, Yuko; Nagasawa, Masahiro; Medina, Johan; Kojima, Itaru

    2015-01-01

    Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca2+ concentration ([Ca2+]c); glucose evoked an immediate elevation of [Ca2+]c, which was followed by a decrease in [Ca2+]c, and after a certain lag period it induced large oscillatory elevations of [Ca2+]c. Initial rapid peak and subsequent reduction of [Ca2+]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca2+, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca2+]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor. PMID:26630567

  15. Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

    Energy Technology Data Exchange (ETDEWEB)

    Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting, E-mail: BTZhu@kumc.edu

    2012-07-15

    Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

  16. T cells play an essential role in anti-F1 mediated rapid protection against bubonic plague.

    Science.gov (United States)

    Levy, Yinon; Flashner, Yehuda; Tidhar, Avital; Zauberman, Ayelet; Aftalion, Moshe; Lazar, Shirley; Gur, David; Shafferman, Avigdor; Mamroud, Emanuelle

    2011-09-16

    Plague, which is initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated. Antibiotics are an effective plague therapy, but antibiotic-resistant Y. pestis strains have been reported and therefore alternative countermeasures are needed. In the present study, we assessed the potential of an F1 plus LcrV-based vaccine to provide protection shortly pre- or post-exposure to a lethal Y. pestis infection. Mice vaccinated up to one day before or even several hours after subcutaneous challenge were effectively protected. Mice immunized one or three days pre-challenge were protected even though their anti-F1 and anti-LcrV titers were below detection levels at the day of challenge. Moreover, using B-cell deficient μMT mice, we found that rapidly induced protective immunity requires the integrity of the humoral immune system. Analysis of the individual contributions of vaccine components to protection revealed that rF1 is responsible for the observed rapid antibody-mediated immunity. Applying anti-F1 passive therapy in the mouse model of bubonic plague demonstrated that anti-F1 F(ab')(2) can delay mortality, but it cannot provide long-lasting protection, as do intact anti-F1 molecules. Fc-dependent immune components, such as the complement system and (to a lesser extent) neutrophils, were found to contribute to mouse survival. Interestingly, T cells but not B cells were found to be essential for the recovery of infected animals following passive anti-F1 mediated therapy. These data extend our understanding of the immune mechanisms required for the development of a rapid and effective post-exposure therapy against plague. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Single-cell pH imaging and detection for pH profiling and label-free rapid identification of cancer-cells.

    Science.gov (United States)

    Hou, Hui; Zhao, Yangyang; Li, Chuanping; Wang, Minmin; Xu, Xiaolong; Jin, Yongdong

    2017-05-11

    Single-cell pH-sensing and accurate detection and label-free fast identification of cancer-cells are two long-standing pursuits in cell and life science, as intracellular pH plays a crucial role in many cellular events and fates, while the latter is vital for early cancer theranostics. Numerous methods based on functionalized nanoparticles and fluorescence probes have been developed for cell pH-sensing, but are often hindered for single-cell studies by their main drawbacks of complicated probe preparation and labeling, low sensitivity and poor reproducibility. Here we report a simple and reliable method for single-cell pH imaging and sensing by innovative combined use of UV-Vis microspectroscopy and common pH indicators. Accurate and sensitive pH detection on single-cell or sub-cell level with good reproducibility is achieved by the method, which enables facile single-cell pH profiling and label-free rapid identification of cancer-cells (due to distinguishable intracellular pH levels) for early cancer diagnosis, and may open a new avenue for pH-related single-cell studies.

  18. A rapid method for assessment of natural killer cell function after multiple receptor crosslinking.

    Science.gov (United States)

    Al-Hubeshy, Z B; Coleman, A; Nelson, M; Goodier, M R

    2011-03-07

    NK cell function is regulated by the integration of signals from activating and inhibitory receptors. We developed an assay to study the effect of co-crosslinking NK cell receptors in pair-wise combinations without the need to purify NK cells. Monoclonal antibodies recognising inhibitory and activating receptors were coated to flat bottomed tissue culture plates and degranulation was measured within unfractionated, freshly isolated resting or cytokine activated peripheral blood mononuclear cells by flow cytometric analysis of CD107a expression. Measured degranulation responses were NK cell specific, since no expression of CD107a was induced in gated T cells. We detected enhancement of degranulation in response to combinations of antibodies against activating NK cell receptors, including CD16, NKG2D, NKp30 and NKp46 compared to each antibody when combined with an isotype matched control antibody. Co-crosslinking of NKG2A resulted in the inhibition of degranulation measured in response to anti-NKp30 or anti-NKp46 alone in both resting or cytokine pre-activated NK cells, but had no effect on CD16 or NKG2D mediated responses. Interferon gamma production was assayed by intracellular cytokine staining and in cell culture supernatants after receptor crosslinking. No IFN-γ could be detected from resting NK cells after receptor crosslinking whereas the pattern of IFN-γ production in cytokine pre-activated NK cells reflected that observed for degranulation. We conclude that this assay is suitable for the analysis of the impact of NK cell receptor co-crosslinking on multiple NK cell functions and has the potential for application to pathologic conditions where limited numbers of cells are available for study. Published by Elsevier B.V.

  19. Rapid effects of 17beta-estradiol on TRPV5 epithelial Ca2+ channels in rat renal cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2009-08-01

    The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca(2+) entry step in transcellular Ca(2+) re-absorption in the kidney. The regulation of transmembrane Ca(2+) flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD(2)) cells at mRNA and protein levels. We demonstrate that 17beta-estradiol (E(2)) is involved in the regulation of Ca(2+) influx in these cells via the epithelial Ca(2+) channels TRPV5. By combining whole-cell patch-clamp and Ca(2+)-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20-50nM E(2) rapidly (<5min) induced a transient increase in inward whole-cell currents and intracellular Ca(2+) via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E(2) in kidney cells. Furthermore, the results suggest calcitropic effects of E(2). The results are discussed in relation to present concepts of non-genomic actions of E(2) in Ca(2+) homeostasis.

  20. Rapid Endolysosomal Escape and Controlled Intracellular Trafficking of Cell Surface Mimetic Quantum-Dots-Anchored Peptides and Glycopeptides.

    Science.gov (United States)

    Tan, Roger S; Naruchi, Kentaro; Amano, Maho; Hinou, Hiroshi; Nishimura, Shin-Ichiro

    2015-09-18

    A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanoparticle-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems.

  1. Cell-on-hydrogel platform made of agar and alginate for rapid, low-cost, multidimensional test of antimicrobial susceptibility.

    Science.gov (United States)

    Sun, Han; Liu, Zhengzhi; Hu, Chong; Ren, Kangning

    2016-08-02

    Antimicrobial resistance (AMR) is a rapidly increasing threat to the effective treatment of infectious diseases worldwide. The two major remedies include: (1) using narrow-spectrum antibiotics based on rapid diagnosis; and (2) developing new antibiotics. A key part of both remedies is the antimicrobial susceptibility test (AST). However, the current standard ASTs that monitor colony formation are costly and time-consuming and the new strategies proposed are not yet practical to be implemented. Herein, we report a strategy to fabricate whole-hydrogel microfluidic chips using alginate-doped agar. This agar-based microfabrication makes it possible to prepare inexpensive hydrogel devices, and allows a seamless link between microfluidics and conventional agar-based cell culture. Different from common microfluidic systems, in our system the cells are cultured on top of the device, similar to normal agar plate culture; on the other hand, the microfluidic channels inside the hydrogel allow precise generation of linear gradient of drugs, thus giving a better performance than the conventional disk diffusion method. Cells in this system are not exposed to any shear flow, which allows the reliable tracking of individual cells and AST results to be obtained within 2-3 hours. Furthermore, our system could test the synergistic effect of drugs through two-dimensional gradient generation. Finally, the platform could be directly implemented to new drug discovery and other applications wherein a fast, cost-efficient method for studying the response of microorganisms upon drug administration is desirable.

  2. Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

    Science.gov (United States)

    Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K

    2017-12-05

    The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils

  3. Rapid preparation and single-cell analysis of concentrated blood smears using a high-throughput blood cell separator and a microfabricated grid film.

    Science.gov (United States)

    You, Dongwon; Oh, Sein; Kim, Byeongyeon; Hahn, Young Ki; Choi, Sungyoung

    2017-07-21

    Cytological examination of peripheral white blood cells inhomogeneously distributed on a blood smear is currently limited by the low abundance and random sampling of the target cells. To address the challenges, we present a new approach to prepare and analyze concentrated blood smears by rapidly enriching white blood cells up to 32-fold with 92% recovery on average at a high throughput (1mL/min) using a deterministic migration-based separator and by systematically analyzing a large number of the cells distributed over a blood slide using a microfabricated grid film. We anticipate that our approach will improve the clinical utility of blood smear tests, while offering the capability to detect rare cell populations. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. A rapid chemical method for lysing Arabidopsis cells for protein analysis

    OpenAIRE

    Takano Tetsuo; Liu Shenkui; Tsugama Daisuke

    2011-01-01

    Abstract Background Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once. Results We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ ...

  5. Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism

    Science.gov (United States)

    Escarmís, Cristina; Carrillo, Elisa C.; Ferrer, Marcela; Arriaza, Juan F. García; Lopez, Nora; Tami, Cecilia; Verdaguer, Nuria; Domingo, Esteban; Franze-Fernández, Maria T.

    1998-01-01

    With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C3Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C3Arg/85 infection. Infection of BHK-Rb cells with C3Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C3-Rb. The selected C3-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9→Ala in VP3 and either Gly-110→Arg or His-108→Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C3Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells. PMID:9811758

  6. Rapid generation of NY-ESO-1-specific CD4(+) THELPER1 cells for adoptive T-cell therapy.

    Science.gov (United States)

    Kayser, Simone; Boβ, Cristina; Feucht, Judith; Witte, Kai-Erik; Scheu, Alexander; Bülow, Hans-Jörg; Joachim, Stefanie; Stevanović, Stefan; Schumm, Michael; Rittig, Susanne M; Lang, Peter; Röcken, Martin; Handgretinger, Rupert; Feuchtinger, Tobias

    2015-05-01

    Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4(+), IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4(+) TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4(+) T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8(+) T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4(+) T cells showed strong specific TH1-responses with IFNγ(+), TNFα(+), IL-2(+) and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4(+) TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients.

  7. Rapid generation of NY-ESO-1-specific CD4+ THELPER1 cells for adoptive T-cell therapy

    Science.gov (United States)

    Kayser, Simone; Boβ, Cristina; Feucht, Judith; Witte, Kai-Erik; Scheu, Alexander; Bülow, Hans-Jörg; Joachim, Stefanie; Stevanović, Stefan; Schumm, Michael; Rittig, Susanne M; Lang, Peter; Röcken, Martin; Handgretinger, Rupert; Feuchtinger, Tobias

    2015-01-01

    Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4+, IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4+ TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4+ T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8+ T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4+ T cells showed strong specific TH1-responses with IFNγ+, TNFα+, IL-2+ and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4+ TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients. PMID:26155389

  8. Development of a Rapid Cell-free Method for Cytotoxicity Assessment of Vapor Phase of Cigarette Smoke

    Directory of Open Access Journals (Sweden)

    Cahours X

    2014-12-01

    Full Text Available Currently, several in vitro tests are widely used to measure toxicological properties of mainstream smoke (Neutral Red Uptake Assay, Micronucleus assay, Ames Test. These tests are necessary to assess cytotoxicity, genotoxicity, and mutagenicity, but are time consuming. This is essentially due to the preparation and the handling of cells. It is difficult to use these in vitro tests as screening method for product testing and development. For a better assessment of the cytotoxicity of the vapor phase, a rapid cell-free method has been developed. This paper describes a capillary electrophoresis cell-free method, based on the depletion of an anti-oxidant L-gamma-glutamyl-L-cysteinylglycine (GSH, applied to an aliquot of vapor phase phosphate buffered saline (PBS-trapped cigarette smoke (as recommended for in vitro testing. The correlation between this method and the survival/viability test (Neutral Red cytotoxicity is excellent (coefficient of correlation (r = 0.99.

  9. Rapid diagnosis of M.tuberculosis meningitis by enumeration of cerebrospinal fluid antigen-specific T cells

    Science.gov (United States)

    Thomas, MM; Hinks, TSC; Raghuraman, S; Ramalingam, N; Ernst, M; Nau, R; Lange, C; Kösters, K; Gnanamuthu, C; John, GT; Marshall, B; Lalvani, A

    2009-01-01

    SUMMARY Setting Hospital in-patients with suspected tuberculous meningitis predominantly in India. Objective To determine whether interferon-γ-secreting Mycobacterium tuberculosis-antigen-specific T cells are present in the cerebrospinal fluid of patients with tuberculous meningitis, and to evaluate the feasibility of cerebrospinal fluid enzyme-linked immunospot for the diagnosis of active tuberculous meningitis. Design Prospective, blinded, hospital-based study. Results The overnight enzyme-linked immunospot assay detected Mycobacterium tuberculosis-antigen-specific interferon-γ-secreting T cells in cerebrospinal fluid from 9 out of 10 prospectively recruited patients with tuberculous meningitis, and 0 out of 7 control patients with meningitis of other aetiology. This corresponds to a diagnostic sensitivity of 90% (95%CI 56-100%) and specificity of 100% (95%CI 59-100%). Conclusion This pilot study demonstrates proof-of-principle for a new T cell-based diagnostic test for tuberculous meningitis which is rapid, sensitive and specific. PMID:18492332

  10. The Rapid Detection of Single Bacterial Cells by Deep UV Micro Raman Spectroscopy.

    Science.gov (United States)

    1992-04-01

    developed for the purpose of rapid bacterial detection. Techniques include mass spectroscopy and its various combinations with chromatography and pyrolysis...Methods: Chromatography and Mass Spectroscopy", Plenum Press, N.Y. 1990. 6. P.J.H. Jackman in "Methods in Microbiology", Vol. 19, eds., R.R., Colwell and R...4847198 issued July 11, 1989. 5. "Ultraviolet Resonance Raman Spectra of Bacteria, Bacterial Spores, Protoplasts and Calcium Dipicolinate", R

  11. Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide.

    Science.gov (United States)

    Watkins, Catherine L; Schmaljohann, Dirk; Futaki, Shiroh; Jones, Arwyn T

    2009-05-13

    The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 microM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the suspension cells studied, but a 10-fold higher concentration was required to observe similar results in adherent cells. At 37 degrees C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of a requirement for nucleation zones to promote this effect. Cholesterol depletion with methyl-beta-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 degrees C, but decreased overall peptide accumulation. Under the same conditions in adherent cells this agent had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide at 4 degrees C in KG1a cells, but this effect could be reversed by re-addition of cholesterol as methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially at low temperatures, suggesting that this feature could be exploited for delivering bioactive entities.

  12. Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration.

    Science.gov (United States)

    Assani, Akym; Moundanga, Sylvie; Beney, Laurent; Gervais, Patrick

    2009-12-01

    Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium. Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration-rehydration. The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock. It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.

  13. Case series: Rapidly growing squamous cell carcinoma after cutaneous surgical intervention

    Directory of Open Access Journals (Sweden)

    Abdullah Ibrahim

    2017-12-01

    This case series explores the various mechanisms of de-novo squamous cell carcinoma development in areas of cutaneous surgical intervention, including graft harvest. It also provides recommendations regarding the necessary precautions to avoid implantation of squamous cell carcinoma into distant sites. Lastly it highlights the importance of surveillance for any suspicious lesions arising from areas of previous cutaneous surgical intervention.

  14. Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41

    NARCIS (Netherlands)

    Vervelde, L.; Matthijs, M.G.R.; van Haarlem, D.A.; de Wit, Sjaak; Jansen, C.A.

    2013-01-01

    Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation

  15. Digital divide research, achievements and shortcomings

    NARCIS (Netherlands)

    van Dijk, Johannes A.G.M.

    2006-01-01

    From the end of the 1990s onwards the digital divide, commonly defined as the gap between those who have and do not have access to computers and the Internet, has been a central issue on the scholarly and political agenda of new media development. This article makes an inventory of 5 years of

  16. Democratisation and Conflict in Ethnically Divided Societies

    NARCIS (Netherlands)

    Vorrath, Judith; Krebs, Lutz

    2009-01-01

    This article reviews three important factors in the academic debate on ethnic civil wars: the role of ethnicity in causing and structuring violence, the spread of ethnic civil wars once they have started, and the influence of democratic transitions in divided societies. The review displays the range

  17. Divide and Multiply: Baptist Diversity in Appalachia.

    Science.gov (United States)

    Dorgan, Howard

    1996-01-01

    Baptists' propensity for splitting apart arises from the faith's avowed love of theological argument. Gives an overview of Baptist denominations, and identifies the main issues that divide them: atonement, predestination, the nature and origins of good and evil, worship practices, church governance, gender issues, and other social and cultural…

  18. Bridging the digital divide with mobile services

    CERN Document Server

    Yelton, Andromeda

    2013-01-01

    In this issue of Library Technology Reports, Andromeda Yelton shows how libraries can build on the breadth of this population to help bridge the digital divide and provide even greater access to information. Yelton breaks down the demographics of mobile internet users, provides examples of how different libraries are reaching out to these populations, and suggests what the future may hold for this trend.

  19. Project DIVIDE Instrument Development. Technical Report # 0810

    Science.gov (United States)

    Ketterlin-Geller, Leanne; Jung, Eunju; Geller, Josh; Yovanoff, Paul

    2008-01-01

    In this technical report, we describe the development of cognitive diagnostic test items that form the basis of the diagnostic system for Project DIVIDE (Dynamic Instruction Via Individually Designed Environments). The construct underlying the diagnostic test is division of fractions. We include a description of the process we used to identify the…

  20. TOWARDS BRIDGING ETHNIC AND RELIGIOUS DIVIDES IN ...

    African Journals Online (AJOL)

    NGOZI

    claim both common ancestry and a common cultural tradition”(p. 218). ... foundation. Among several other reasons that results to ethno- religious divide in Nigeria is the amalgamation event which is the hand work of the European imperial powers under the ... ethnicity is said to be rooted in the very set up of Nigeria,but it got.

  1. A Portable Cell Maintenance System for Rapid Toxicity Monitoring Final Report CRADA No. TC-02081-04

    Energy Technology Data Exchange (ETDEWEB)

    Kane, S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Zhou, P. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2017-09-27

    The Phase I STTR research project was targeted at meeting the objectives and requirements stated in STTR solicitation A04-T028 for a Portable Cell Maintenance System for Rapid Toxicity Monitoring. In accordance with the requirements for STTR programs, collaboration was formed between a small business, Kionix, Inc., and The Regents of the University of California, Lawrence Livermore National Laboratory (LLNL). The collaboration included CytoDiscovery, Inc. (CDI) which, in collaboration with Kionix, provided access to membrane chip technology and provided program support and coordination. The objective of the overall program (excerpted from the original solicitation) was: “To develop a small, portable cell maintenance system for the transport, storage, and monitoring of viable vertebrate cells and tissues.” The goal of the Phase I project was to demonstrate the feasibility of achieving the program objectives utilizing a system comprised of a small-size, microfluidic chip-based cell maintenance cartridge (CMC) and a portable cell maintenance system (CMS) capable of housing a minimum of four CMCs. The system was designed to be capable of optimally maintaining multiple vertebrate cell types while supporting a wide variety of cellular assays.

  2. Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

    Science.gov (United States)

    Richel, D J; Johnsen, H E; Canon, J; Guillaume, T; Schaafsma, M R; Schenkeveld, C; Hansen, S W; McNiece, I; Gringeri, A J; Briddell, R; Ewen, C; Davies, R; Freeman, J; Miltenyi, S; Symann, M

    2000-02-01

    The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

  3. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Directory of Open Access Journals (Sweden)

    Yin Zongyi

    Full Text Available Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods. Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets. In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  4. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Science.gov (United States)

    Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li

    2017-01-01

    Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  5. Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

    Directory of Open Access Journals (Sweden)

    Kathrin Davari

    2017-04-01

    Full Text Available Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP, and RNA polymerase II (RNA Pol II ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD and activation of the positive transcription elongation factor (pTEFb. Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation.

  6. A combination of four cell types for rapid detection of enteroviruses in clinical specimens.

    Science.gov (United States)

    Dagan, R; Menegus, M A

    1986-07-01

    Isolation in cell culture is currently the most sensitive and reliable way to demonstrate enterovirus (EV) in clinical specimens. During July-October 1982 and 1983, we studied the impact of adding two new cell lines, Buffalo green monkey kidney (BGM) and human rhabdomyosarcoma (RD), to the more traditional cell combination used for EV isolation, human embryonic lung (HEL) and primary cynomolgus monkey kidney (CMK) cells; 2,558 specimens were studied: 632 fecal, 677 respiratory, 537 CSF, and 712 blood. An EV was isolated from 417 (16%); of these, 77 (18%) were positive only in BGM or RD; 35% (146/417) of the specimens were positive in BGM, RD, or both, at least one day earlier than in the traditional cells. BGM cells were helpful in isolation of group B coxsackieviruses (CB): 99% of 121 positive specimens were detected in BGM vs 73% in CMK and 23% in HEL; 72% of the CB isolates were detected by day 2 in BGM vs 48% in CMK and 0% in HEL. RD cells were helpful in the isolation of echoviruses: 59% of the 189 positive specimens were detected in RD vs 67% in HEL and 58% in CMK. RD was the only positive cell type in 28/189 (15%) positive specimens; 31% of the echovirus isolates were detected by day 2 in RD, vs 20% in HEL and 19% in CMK. Using the cell types described, we provided the clinician with results in 42% of the EV-positive specimens by day 2 after inoculation and in 61% by day 4.

  7. Conquering the digital divide: Botswana and South Korea digital divide status and interventions

    Directory of Open Access Journals (Sweden)

    Nonofo C. Sedimo

    2011-11-01

    Objectives: Bridging the digital divide and narrowing the intra-national divide brings about global information and communication technology (ICT usage that translates into changing work patterns and eventually transformed economies. This article outlines the different interventions implemented in Botswana to bridge the divide. The South Korean experience in bridging the divide is discussed so as to serve as lessons on how to effectively bridge the divide to Botswana’s initiatives. Method: Using a mix of exploratory and empirical study, this article presents the findings on the status of ICT uptake in Botswana and investigates the level of the digital divide in the country. Results: The results of the study show that the digital divide is much more evident in Botswana than in South Korea. South Korea has put in place robust strategic initiatives towards reducing the digital divide and this has largely transcended into its transformation into a full-fledged knowledge society. Conclusion: This article is timely as it unearths the different pointers that may be utilised in policy formation and what interventions need to be taken at both the individual and national level to bridge the digital divide.

  8. Conquering the digital divide: Botswana and South Korea digital divide status and interventions

    Directory of Open Access Journals (Sweden)

    Nonofo C. Sedimo

    2011-03-01

    Full Text Available Background: Botswana is putting in place initiatives towards establishing itself as a knowledgebased economy. Transformation from a resource-based to a knowledge-based economy is partly hinged on innovation, research and development capability, knowledge channels, and the funding of research and development activities.Objectives: Bridging the digital divide and narrowing the intra-national divide brings about global information and communication technology (ICT usage that translates into changing work patterns and eventually transformed economies. This article outlines the different interventions implemented in Botswana to bridge the divide. The South Korean experience in bridging the divide is discussed so as to serve as lessons on how to effectively bridge the divide to Botswana’s initiatives.Method: Using a mix of exploratory and empirical study, this article presents the findings on the status of ICT uptake in Botswana and investigates the level of the digital divide in the country.Results: The results of the study show that the digital divide is much more evident in Botswana than in South Korea. South Korea has put in place robust strategic initiatives towards reducing the digital divide and this has largely transcended into its transformation into a full-fledged knowledge society.Conclusion: This article is timely as it unearths the different pointers that may be utilised in policy formation and what interventions need to be taken at both the individual and national level to bridge the digital divide.

  9. Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Morotomi, M.; Ohno, T.; Mutai, M.

    1988-05-01

    A dot blot hybridization procedure with /sup 32/P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.

  10. A new aggregation-induced emission fluorescent probe for rapid detection of nitroreductase and its application in living cells

    Science.gov (United States)

    Xu, Gaoping; Tang, Yonghe; Ma, Yanyan; Xu, An; Lin, Weiying

    2018-01-01

    The biological activity of nitroreductase (NTR) is closely related to biological hypoxia status in organisms. The development of effective methods for monitoring the activity of NTR is of great significance for medical diagnosis and tumor research. Toward this goal, we have developed a new aggregation-induced emission (AIE) fluorescence NTR probe TPE-HY used the tetraphenylethene as the fluorophore, and used the nitro group as the NTR recognition site. The probe TPE-HY has many excellent properties, including rapid response, AIE characteristics, high sensitivity and selectivity, and low cytotoxicity. Importantly, the probe TPE-HY is successfully applied to monitor endogenous NTR in living HeLa cells.

  11. Tetracycline rapidly reaches all the constituent cells of uropathogenic Escherichia coli biofilms

    Science.gov (United States)

    Stone, G.; Wood, P.; Dixon, L.; Keyhan, M.; Matin, A.; Demain, A. L. (Principal Investigator)

    2002-01-01

    We have developed a method for visualizing Escherichia coli cells that are exposed to tetracycline in a biofilm, based on a previous report that liposomes containing the E. coli TetR(B) protein fluoresce when exposed to this antibiotic. By our method, cells devoid of TetR(B) also exhibited tetracycline-dependent fluorescence. At 50 microg of tetracycline ml(-1), planktonic cells of a uropathogenic E. coli (UPEC) strain developed maximal fluorescence after 7.5 to 10 min of exposure. A similar behavior was exhibited by cells in a 24- or 48-h UPEC biofilm, as examined by confocal laser microscopy, regardless of whether they lined empty spaces or occupied densely packed regions. Further, a comparison of phase-contrast and fluorescent images of corresponding biofilm zones showed that all the cells fluoresced. Thus, all the biofilm cells were exposed to tetracycline and there were no pockets within the biofilm where the antibiotic failed to reach. It also appeared unlikely that niches of reduced exposure to the antibiotic existed within the biofilms.

  12. Rapid and unambiguous detection of DNase I hypersensitive site in rare population of cells.

    Directory of Open Access Journals (Sweden)

    Wei-Ping Zeng

    Full Text Available DNase I hypersensitive (DHS sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×10(4 cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.

  13. Rapid single-step induction of functional neurons from human pluripotent stem cells.

    Science.gov (United States)

    Zhang, Yingsha; Pak, Changhui; Han, Yan; Ahlenius, Henrik; Zhang, Zhenjie; Chanda, Soham; Marro, Samuele; Patzke, Christopher; Acuna, Claudio; Covy, Jason; Xu, Wei; Yang, Nan; Danko, Tamas; Chen, Lu; Wernig, Marius; Südhof, Thomas C

    2013-06-05

    Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Rapid Isolation of Monoclonal Antibodies Specific for Cell Surface Differentiation Antigens

    Science.gov (United States)

    Barclay, Stephen L.; Smith, Alan M.

    1986-06-01

    Two immunization procedures were compared for their ability to yield monoclonal antibodies that react with plasma membrane-bound differentiation antigens of Dictyostelium. In the first method, hybridomas prepared from BALB/c mice immunized with aggregating amoebae produced monoclonal antibodies that recognized antigens present on both growing and aggregating Dictyostelium amoebae. None of the monoclonal antibodies reacted with only the injected aggregation-stage cell type. In contrast, monoclonal antibodies that reacted with differentiation antigens were easily obtained by primary immunization of BALB/c mice with living aggregation-stage cells, followed by secondary immunization with a preparation of plasma membrane from aggregating cells or intact aggregating cells mixed with polyclonal BALB/c antiserum raised against undifferentiated cells. By this method, approximately 20% of all anti-Dictyostelium monoclonal antibodies obtained in a fusion are specific for differentiation antigens. The properties and developmental regulation of several of these antigens are described. The possible uses of this immunological method to detect unique determinants on other kinds of cells and the likely immune mechanisms that make it successful are discussed.

  15. Herpes simplex virus type 2 induces rapid cell death and functional impairment of murine dendritic cells in vitro

    NARCIS (Netherlands)

    Jones, CA; Fernandez, M; Herc, K; Bosnjak, L; Miranda-Saksena, M; Boadle, RA; Cunningham, A

    2003-01-01

    Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h

  16. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

    Directory of Open Access Journals (Sweden)

    Radovan Fiser

    Full Text Available Bordetella adenylate cyclase toxin-hemolysin (CyaA penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

  17. Human Adipose-Derived Stem Cells on Rapid Prototyped Three-Dimensional Hydroxyapatite/Beta-Tricalcium Phosphate Scaffold.

    Science.gov (United States)

    Canciani, Elena; Dellavia, Claudia; Ferreira, Lorena Maria; Giannasi, Chiara; Carmagnola, Daniela; Carrassi, Antonio; Brini, Anna Teresa

    2016-05-01

    In the study, we assess a rapid prototyped scaffold composed of 30/70 hydroxyapatite (HA) and beta-tricalcium-phosphate (β-TCP) loaded with human adipose-derived stem cells (hASCs) to determine cell proliferation, differentiation toward osteogenic lineage, adhesion and penetration on/into the scaffold.In this in vitro study, hASCs isolated from fat tissue discarded after plastic surgery were expanded, characterized, and then loaded onto the scaffold. Cells were tested for: viability assay (Alamar Blue at days 3, 7 and Live/Dead at day 32), differentiation index (alkaline phosphatase activity at day 14), scaffold adhesion (standard error of the mean analysis at days 5 and 18), and penetration (ground sections at day 32).All the hASC populations displayed stemness markers and the ability to differentiate toward adipogenic and osteogenic lineages.Cellular vitality increased between 3 and 7 days, and no inhibitory effect by HA/β-TCP was observed. Under osteogenic stimuli, scaffold increased alkaline phosphatase activity of +243% compared with undifferentiated samples. Human adipose-derived stem cells adhered on HA/β-TCP surface through citoplasmatic extensions that occupied the macropores and built networks among them. Human adipose derived stem cells were observed in the core of HA/β-TCP. The current combination of hASCs and HA/β-TCP scaffold provided encouraging results. If authors' data will be confirmed in preclinical models, the present engineering approach could represent an interesting tool in treating large bone defects.

  18. A portable cell-based optical detection device for rapid detection of Listeria and Bacillus toxins

    Science.gov (United States)

    Banerjee, Pratik; Banada, Padmapriya P.; Rickus, Jenna L.; Morgan, Mark T.; Bhunia, Arun K.

    2005-11-01

    A mammalian cell-based optical biosensor was built to detect pathogenic Listeria and Bacillus species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic L. monocytogenes strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from B. cereus strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic L. innocua (F4247) and a B. substilis induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic Listeria and Bacillus species and can potentially be used onsite for food safety or in biosecurity application.

  19. Analyzing Broadband Divide in the Farming Sector

    DEFF Research Database (Denmark)

    Jensen, Michael; Gutierrez Lopez, Jose Manuel; Pedersen, Jens Myrup

    2013-01-01

    , upstream and downstream connection. The main constraint is that farms are naturally located in rural areas where the required access broadband data rates are not available. This paper studies the broadband divide in relation to the Danish agricultural sector. Results show how there is an important......Agriculture industry has been evolving for centuries. Currently, the technological development of Internet oriented farming tools allows to increase the productivity and efficiency of this sector. Many of the already available tools and applications require high bandwidth in both directions...... difference between the broadband availability for farms and the rest of the households/buildings the country. This divide may be slowing down the potential technological development of the farming industry, in order to keep their competitiveness in the market. Therefore, broadband development in rural areas...

  20. REGENERATIVE GAS TURBINES WITH DIVIDED EXPANSION

    DEFF Research Database (Denmark)

    Elmegaard, Brian; Qvale, Einar Bjørn

    2004-01-01

    their efficiency are always of interest. Recently, two independent studies have proposed recuperated gas turbines to be configured with the turbine expansion divided, in order to obtain higher efficiency. The idea is to operate the system with a gas generator and a power turbine, and use the gas from the gas......Recuperated gas turbines are currently drawing an increased attention due to the recent commercialization of micro gas turbines with recuperation. This system may reach a high efficiency even for the small units of less than 100 kW. In order to improve the economics of the plants, ways to improve...... divided expansion can be advantageous under certain circumstances. But, in order for todays micro gas turbines to be competitive, the thermodynamic efficiencies will have to be rather high. This requires that all component efficiencies including the recuperator effectiveness will have to be high...

  1. Cellular forces : adhering, shaping, sensing and dividing

    NARCIS (Netherlands)

    Hoorn, Hedde van

    2014-01-01

    Life’s building block is a cell. Different cell types are differentiated by specific functional properties. A white blood cell, for instance, can get rid of bacteria and many muscle cells contract together for proper muscle function. Deformation and force exertion play important roles in these

  2. A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections.

    Directory of Open Access Journals (Sweden)

    Matthias Eberl

    2009-02-01

    Full Text Available Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL-6, interferon (IFN-gamma, tumor necrosis factor (TNF-alpha, and oncostatin M (OSM; the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL. Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+ effector alphabeta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe

  3. Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV.

    Science.gov (United States)

    Rodriguez-Garcia, M; Shen, Z; Barr, F D; Boesch, A W; Ackerman, M E; Kappes, J C; Ochsenbauer, C; Wira, C R

    2017-03-01

    Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c + DC subsets were identified: CD11b + and CD11b low DCs. CD11b + CD14 + DCs were the most abundant throughout the tract. A majority of CD11c + CD14 + cells corresponded to CD1c + myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103 + DCs, located exclusively in the endometrium, whereas DC-SIGN + DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14 + DC-SIGN + as well as CD14 + DC-SIGN - cells captured virus, with ∼30% of these cells representing CD1c + myeloid DCs. CD103 + DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.

  4. Polymer based biosensor for rapid electrochemical detection of virus infection of human cells

    DEFF Research Database (Denmark)

    Kiilerich-Pedersen, Katrine; Poulsen, Claus R.; Jain, Titoo

    2011-01-01

    The demand in the field of medical diagnostics for simple, cost efficient and disposable devices is growing. Here, we present a label free, all-polymer electrochemical biosensor for detection of acute viral disease. The dynamics of a viral infection in human cell culture was investigated in a micro...... fluidic system on conductive polymer PEDOT:TsO microelectrodes by electrochemical impedance spectroscopy and video time lapse microscopy.Employing this sensitive, real time electrochemical technique, we could measure the immediate cell response to cytomegalovirus, and detect an infection within 3h, which...

  5. Measurement of rapid membrane permeation in cell suspensions by application of a generalized capillary method

    DEFF Research Database (Denmark)

    Klösgen, Beate; Schönert, Hansjürgen; Deuticke, Bernhard

    1988-01-01

    the diffusion process of a solute in a composite system was derived using a series-parallel-pathway model with explicit consideration of the diffusion pathways inside and between the cells. This renders the technique insensitive to unstirred layer effects. Any single cell population of known size distribution...... may be investigated. High permeabilities (above 5 · 10-3 cm/s) can be measured with the greatest precision, but lower permeabilities, down to a limit of about 5 · 10-4 cm/s, may also be determined by the method. Measurements in erythrocyte suspensions have been made using non...... of suspensions of membrane vesicles....

  6. Rapid and label-free cell detection by metal-cluster-decorated carbon nanotube biosensors.

    Science.gov (United States)

    Ishikawa, Fumiaki N; Stauffer, Beth; Caron, David A; Zhou, Chongwu

    2009-06-15

    In this paper, the use of carbon nanotube biosensors toward alga cell detection was examined. The biosensor devices were fabricated on complete 4 in. wafers by first growing carbon nanotubes (CNTs) and then depositing metal electrodes using a shadow mask. In addition, we decorated the biosensors with metal-clusters resulted in enhancing the sensitivity by 2000-folds and has enabled the detection of streptavidin down to 10 pM concentration. This sensitivity enhancement was attributed to activation of CNT channels due to formation of Schottky junctions between CNTs and metal-clusters. Real-time cell detection has been successfully carried out using the CNT biosensors for two kinds of alga related to brown tides: Aureococcus anophagefferens and BT3. Functionalization of the CNT biosensors with the monoclonal antibody for A. anophagefferens has led to detection at a concentration of 10(4) cells/ml, with sensitivity lower than 10(4) cells/ml projected based on the signal-to-noise ratio of the sensors. Further functionalization with tween 20 led to suppression of non-specific binding of BT3 and enabled label-free and selective detection of A. anophagefferens. These nanobiosensors may find potential applications for environmental monitoring and disease diagnosis.

  7. Integrated Systems for Rapid Point of Care (PoC) Blood Cell Analysis

    NARCIS (Netherlands)

    Van Berkel, C.; Gwyer, J.D.; Deane, S.; Hollis, V.; Holloway, J.; Green, N.; Morgan, H.M.

    2011-01-01

    Counting the different subpopulations of cells in a fingerprick of human blood is important for a number of clinical Point of Care applications. It is a challenge to demonstrate the integration of sample preparation and detection techniques in a single platform. In this article we review the

  8. Rapid desensitization of the histamine H2 receptor on the human monocytic cell line U937

    NARCIS (Netherlands)

    Smit, M J; Leurs, R; Shukrula, S R; Bast, A; Timmerman, H

    1994-01-01

    In the present study we have subjected the histamine H2 receptor on the monocytic cell line U937 to a thorough pharmacological characterization using a series of selective histamine H1, H2 and H3 receptor agonists and antagonists. Recent reports have demonstrated the existence of a histamine H2

  9. Tissue-resident adult stem cell populations of rapidly self-renewing organs

    NARCIS (Netherlands)

    Barker, N.; Bartfeld, S.; Clevers, H.

    2010-01-01

    The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem

  10. Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH

    Energy Technology Data Exchange (ETDEWEB)

    Greulich, K.M.; Rhein, A.P.; Brueckner, M.; Molls, M. [Department of Radiation Oncology, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich (Germany); Kreja, L. [Institute for Occupational, Social and Environmental Medicine, University of Ulm, Ulm (Germany); Heinze, B. [Department of Medical Genetics, University of Ulm, Ulm (Germany); Weier, H.-U.G. [Life Sciences Division, E.O. Lawrence Berkeley National Laboratory, Berkeley, CA (United States); Fuchs, P. [Vysis GmbH, Bergisch-Gladbach (Germany)

    2000-07-20

    Structural chromosome aberrations (SCAs) are sensitive indicators of a preceding exposure of the hematopoietic system to ionizing radiation. Cytogenetic investigations have therefore become routine tools for an assessment of absorbed radiation doses and their biological effects after occupational exposure or radiation accidents. Due to its speed and ease of use, fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes has become a method of choice to visualize SCAs. Until recently, this technique was limited to a rather small number of chromosomes, which could be tested simultaneously. As a result, only a fraction of the structural aberrations present in a sample could be detected and the overall dose effect had to be calculated by extrapolation. The recent introduction of two genome-wide screening techniques in tumor research, i.e., Spectral Karyotyping (SKY) and multicolor FISH (mFISH) now allows the detection of translocations involving any two non-homologous chromosomes. The present study was prompted by our desire to bring the power of mFISH to bear for the rapid identification of radiation-induced SCAs. We chose two model systems to investigate the utility of mFISH: lymphocytes that were exposed in vitro to 3 Gy photons and single hematopoietic progenitor cell colonies isolated from a Chernobyl victim 9 years after in vivo exposure to 5.4 Sv. In lymphocytes, we found up to 15 different chromosomes involved in rearrangements indicating complex radiation effects. Stable aberrations detected in hematopoietic cell colonies, on the other hand, showed involvement of up to three different chromosomes. These results demonstrated that mFISH is a rapid and powerful approach to detect and characterize radiation-induced SCAs in the hemopoietic system. The application of mFISH is expected to result in a more detailed and, thus, more informative picture of radiation effects. Eventually, this technique will allow researchers to rapidly delineate

  11. Hypercytotoxicity and rapid loss of NKp44+ innate lymphoid cells during acute SIV infection.

    Directory of Open Access Journals (Sweden)

    Haiying Li

    2014-12-01

    Full Text Available HIV/SIV infections break down the integrity of the gastrointestinal mucosa and lead to chronic immune activation and associated disease progression. Innate lymphoid cells (ILCs, distinguishable by high expression of NKp44 and RORγt, play key roles in mucosal defense and homeostasis, but are depleted from gastrointestinal (GI tract large bowel during chronic SIV infection. However, less is known about the kinetics of ILC loss, or if it occurs systemically. In acute SIV infection, we found a massive, up to 8-fold, loss of NKp44+ILCs in all mucosae as early as day 6 post-infection, which was sustained through chronic disease. Interestingly, no loss of ILCs was observed in mucosa-draining lymph nodes. In contrast, classical NK cells were not depleted either from gut or draining lymph nodes. Both ILCs and NK cells exhibited significantly increased levels of apoptosis as measured by increased Annexin-V expression, but while classical NK cells also showed increased proliferation, ILCs did not. Interestingly, ILCs, which are normally noncytolytic, dramatically upregulated cytotoxic functions in acute and chronic infection and acquired a polyfunctional phenotype secreting IFN-γ, MIP1-β, and TNF-α, but decreased production of the prototypical cytokine, IL-17. Classical NK cells had less dramatic functional change, but upregulated perforin expression and increased cytotoxic potential. Finally, we show that numerical and functional loss of ILCs was due to increased apoptosis and ROR γt suppression induced by inflammatory cytokines in the gut milieu. Herein we demonstrate the first evidence for acute, systemic, and permanent loss of mucosal ILCs during SIV infection associated with reduction of IL-17. The massive reduction of ILCs involves apoptosis without compensatory de novo development/proliferation, but the full mechanism of depletion and the impact of functional change so early in infection remain unclear.

  12. Rapid induction of orthotopic hepatocellular carcinoma in immune-competent rats by non-invasive ultrasound-guided cells implantation

    Directory of Open Access Journals (Sweden)

    Pan Huay-Ben

    2010-07-01

    Full Text Available Abstract Background The fact that prognoses remain poor in patients with advanced hepatocellular carcinoma highlights the demand for suitable animal models to facilitate the development of anti-cancer medications. This study employed a relatively non-invasive approach to establish an orthotopic hepatocellular carcinoma model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Methods Rat Novikoff hepatoma cells were injected percutaneously into the liver lobes of Sprague-Dawley rats under the guidance of high resolution ultrasound. The implantation rate and the correlation between dissected and ultrasound-measured tumor sizes were evaluated. A similar induction procedure was performed by means of laparotomy in a different group of rats. Pairs of tumor measurement were compared by ultrasound and computerized tomography scan. Rats with a successful establishment of the tumor were divided into the treatment (7-day low-dose epirubicin group and the control group. The tumor sizes were non-invasively monitored by the same ultrasound machine. Blood and tumor tissues from tumor-bearing rats were examined by biochemical and histological analysis respectively. Results Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic hepatocellular carcinoma in 60.4% (55/91 of the Sprague-Dawley rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P Conclusions Ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic hepatocellular carcinoma in Sprague-Dawley rats. Short-term and low-dose epirubicin chemotherapy had perturbed tumor progression by inducing apoptosis and neovascularization blockade.

  13. Rapid Biosynthesis of Silver Nanoparticles Using Pepino (Solanum muricatum Leaf Extract and Their Cytotoxicity on HeLa Cells

    Directory of Open Access Journals (Sweden)

    Mónica Gorbe

    2016-04-01

    Full Text Available Within nanotechnology, gold and silver nanostructures have unique physical, chemical, and electronic properties [1,2], which make them suitable for a number of applications. Moreover, biosynthetic methods are considered to be a safer alternative to conventional physicochemical procedures for both the environmental and biomedical applications, due to their eco-friendly nature and the avoidance of toxic chemicals in the synthesis. For this reason, employing bio routes in the synthesis of functionalized silver nanoparticles (FAgNP have gained importance recently in this field. In the present study, we report the rapid synthesis of FAgNP through the extract of pepino (Solanum muricatum leaves and employing microwave oven irradiation. The core-shell globular morphology and characterization of the different shaped and sized FAgNP, with a core of 20–50 nm of diameter is established using the UV-Visible spectroscopy (UV-vis, field emission scanning electron microscopy (FESEM, transmission electron microscopy (TEM and Zeta potential and dynamic light scanning (DLS studies. Moreover, cytotoxic studies employing HeLa (human cervix carcinoma cells were undertaken to understand FAgNP interactions with cells. HeLa cells showed significant dose dependent antiproliferative activity in the presence of FAgNP at relatively low concentrations. The calculated IC50 value was 37.5 µg/mL, similar to others obtained for FAgNPs against HeLa cells.

  14. Rapidly developed squamous cell carcinoma after laser therapy used to treat chemical burn wound: a case report.

    Science.gov (United States)

    Cho, Hyung-Rok; Kwon, Soon-Sung; Chung, Seum; Kie, Jeong-Hae

    2015-02-07

    In chronic wounds, especially burn scars, malignant tumors can arise. However, it is rare for a subacute burn injury to change to a malignant lesion within one month. Moreover, a case of squamous cell carcinoma arising from HeNe laser therapy after a chemical burn has never been reported. In this report, we examine a rare case of squamous cell carcinoma arising from HeNe laser therapy after a chemical burn. Because pathologic investigations were made from the first operation, both early detection of the squamous cell carcinoma and consideration of the HeNe laser therapy as a risk factor for the skin cancer were possible. The cancer was completely removed and reconstruction of the defect was successfully achieved in a timely manner. Although there has as yet been no reported case of squamous cell carcinoma induced by laser therapy, it is important for clinicians to recognize both the possibility of laser-induced cancer and the rapid change of cancer, so they can provide appropriate and timely treatment.

  15. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ninomiya, Yuichi [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Nishiyama, Masahiko, E-mail: yamacho@saitama-med.ac.jp [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  16. Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

    Science.gov (United States)

    Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

    2003-01-01

    Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

  17. Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidney cell cultures.

    Science.gov (United States)

    Callihan, D R; Menegus, M A

    1984-04-01

    The performance of a culture system for isolation of herpes simplex virus, consisting of one tube each of human embryonic lung fibroblasts and primary rabbit kidney cells, was evaluated. Cultures were incubated at 37 degrees C on a roller drum and observed daily for characteristic cytopathic effect for 5 days. During 1982, a positive isolation rate of 28.1% was seen among 3,154 specimens submitted. Cultures from genital sources were positive more frequently from males (43.8%) than from females (25.5%). Oral lesion cultures were positive as often from males (34.6%) as from females (38.4%). Although detection of herpes simplex virus occurred significantly earlier in rabbit kidney cells on days 1 and 2 of incubation, by day 3 the number of positive cultures was nearly the same in both cell types. By day 4 of incubation, 99.5% of the positive cultures were detected. These results demonstrate that cell culture can be a rapid and sensitive method for detecting herpes simplex virus.

  18. Rapid deterioration of preexisting renal insufficiency after autologous mesenchymal stem cell therapy

    OpenAIRE

    Jun-Seop Kim; Jong-Hak Lee; Owen Kwon; Jang-Hee Cho; Ji-Young Choi; Sun-Hee Park; Chan-Duck Kim; Yong-Jin Kim; Yong-Lim Kim

    2017-01-01

    Administration of autologous mesenchymal stem cells (MSCs) has been shown to improve renal function and histological findings in acute kidney injury (AKI) models. However, its effects in chronic kidney disease (CKD) are unclear, particularly in the clinical setting. Here, we report our experience with a CKD patient who was treated by intravenous infusion of autologous MSCs derived from adipose tissue in an unknown clinic outside of Korea. The renal function of the patient had been stable for ...

  19. Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications

    Directory of Open Access Journals (Sweden)

    Dana M. Cairns

    2016-09-01

    Full Text Available Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.

  20. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn

    2007-01-01

    Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein to that e......Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T...

  1. A system for the rapid detection of bacterial contamination in cell-based therapeutica

    Science.gov (United States)

    Bolwien, Carsten; Erhardt, Christian; Sulz, Gerd; Thielecke, Hagen; Johann, Robert; Pudlas, Marieke; Mertsching, Heike; Koch, Steffen

    2010-02-01

    Monitoring the sterility of cell or tissue cultures is of major concern, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. Our sterility-control system is based on a Raman micro-spectrometer and is able to perform fast sterility testing on microliters of liquid samples. In conventional sterility control, samples are incubated for weeks to proliferate the contaminants to concentrations above the detection limit of conventional analysis. By contrast, our system filters particles from the liquid sample. The filter chip fabricated in microsystem technology comprises a silicon nitride membrane with millions of sub-micrometer holes to retain particles of critical sizes and is embedded in a microfluidic cell specially suited for concomitant microscopic observation. After filtration, identification is carried out on the single particle level: image processing detects possible contaminants and prepares them for Raman spectroscopic analysis. A custom-built Raman-spectrometer-attachment coupled to the commercial microscope uses 532nm or 785nm Raman excitation and records spectra up to 3400cm-1. In the final step, the recorded spectrum of a single particle is compared to an extensive library of GMP-relevant organisms, and classification is carried out based on a support vector machine.

  2. Differentially pumped spray deposition as a rapid screening tool for organic and perovskite solar cells.

    Science.gov (United States)

    Jung, Yen-Sook; Hwang, Kyeongil; Scholes, Fiona H; Watkins, Scott E; Kim, Dong-Yu; Vak, Doojin

    2016-02-08

    We report a spray deposition technique as a screening tool for solution processed solar cells. A dual-feed spray nozzle is introduced to deposit donor and acceptor materials separately and to form blended films on substrates in situ. Using a differential pump system with a motorised spray nozzle, the effect of film thickness, solution flow rates and the blend ratio of donor and acceptor materials on device performance can be found in a single experiment. Using this method, polymer solar cells based on poly(3-hexylthiophene) (P3HT):(6,6)-phenyl C61 butyric acid methyl ester (PC61BM) are fabricated with numerous combinations of thicknesses and blend ratios. Results obtained from this technique show that the optimum ratio of materials is consistent with previously reported values confirming this technique is a very useful and effective screening method. This high throughput screening method is also used in a single-feed configuration. In the single-feed mode, methylammonium iodide solution is deposited on lead iodide films to create a photoactive layer of perovskite solar cells. Devices featuring a perovskite layer fabricated by this spray process demonstrated a power conversion efficiencies of up to 7.9%.

  3. Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates.

    Science.gov (United States)

    Andrews, R G; Briddell, R A; Knitter, G H; Rowley, S D; Appelbaum, F R; McNiece, I K

    1995-01-01

    We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG-CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of

  4. Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR.

    Science.gov (United States)

    Martorell, P; Querol, A; Fernández-Espinar, M T

    2005-11-01

    Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 x 10(5) to 3.8 CFU/ml in sweet wine and from 5 x 10(6) to 50 CFU/ml in red wine.

  5. Understanding the Capsanthin Tails in Regulating the Hydrophilic-Lipophilic Balance of Carbon Dots for a Rapid Crossing Cell Membrane.

    Science.gov (United States)

    Chen, Jing; Zhang, Xiang; Zhang, Ye; Wang, Wei; Li, Shuya; Wang, Yucai; Hu, Mengyue; Liu, Li; Bi, Hong

    2017-10-03

    Here we use natural Chinese paprika to prepare a new kind of amphiphilic carbon dot (A-Dot) that exhibits bright, multicolored fluorescence and contains hydrophilic groups as well as lipophilic capsanthin tails on the surface. It is found that the capsanthin tails in a phospholipid-like structure can promote cell internalization of the A-Dots via crossing cell membranes rapidly in an energy-independent fashion. Compared to highly hydrophilic carbon dots (H-Dots), a control sample prepared from the microwave thermolysis of citric acid and ethylenediamine, our synthesized A-Dots can be taken up by CHO, HeLa, and HFF cells more easily. More importantly, we develop a method to calibrate the hydrophilic-lipophilic balance (HLB) values of various kinds of carbon dots (C-Dots). HLB values of A-Dots and H-Dots are determined to be 6.4 and 18.4, respectively. Moreover, we discover that the cellular uptake efficiency of C-Dots is closely related to their HLBs, and the C-Dots with an HLB value of around 6.4 cross the cell membrane easier and faster. As we regulate the HLB value of the A-Dots from 6.4 to 15.3 by removing the capsanthin tails from their surfaces via alkali refluxing, it is found that the refluxed A-Dots can hardly cross HeLa cell membranes. Our work is an essential step toward understanding the importance of regulating the HLB values as well as the surface polarity of the C-Dots for their practical use in bioimaging and also provides a simple but effective way to judge whether the C-Dots in hand are appropriate for cell imaging.

  6. Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation

    Science.gov (United States)

    2013-01-01

    Background Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood. In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-l-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO2 conditions (ImF-H). Results During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions. Conclusion Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions. PMID:23915242

  7. Rapid production of a H₉ N₂ influenza vaccine from MDCK cells for protecting chicken against influenza virus infection.

    Science.gov (United States)

    Ren, Zhenghua; Lu, Zhongzheng; Wang, Lei; Huo, Zeren; Cui, Jianhua; Zheng, Tingting; Dai, Qing; Chen, Cuiling; Qin, Mengying; Chen, Meihua; Yang, Rirong

    2015-04-01

    H9N2 subtype avian influenza viruses are widespread in domestic poultry, and vaccination remains the most effective way to protect the chicken population from avian influenza pandemics. Currently, egg-based H9N2 influenza vaccine production has several disadvantages and mammalian MDCK cells are being investigated as candidates for influenza vaccine production. However, little research has been conducted on low pathogenic avian influenza viruses (LPAIV) such as H9N2 replicating in mammalian cells using microcarrier beads in a bioreactor. In this study, we present a systematic analysis of a safe H9N2 influenza vaccine derived from MDCK cells for protecting chickens against influenza virus infection. In 2008, we isolated two novel H9N2 influenza viruses from chickens raised in southern China, and these H9N2 viruses were adapted to MDCK cells. The H9N2 virus was produced in MDCK cells in a scalable bioreactor, purified, inactivated, and investigated for use as a vaccine. The MDCK-derived H9N2 vaccine was able to induce high titers of neutralizing antibodies in chickens of different ages. Histopathological examination, direct immunofluorescence, HI assay, CD4(+)/CD8(+) ratio test, and cytokine evaluation indicated that the MDCK-derived H9N2 vaccine evoked a rapid and effective immune response to protect chickens from influenza infection. High titers of H9N2-specific antibodies were maintained in chickens for 5 months, and the MDCK-derived H9N2 vaccine had no effects on chicken growth. The use of MDCK cells in bioreactors for LPAIV vaccine production is an attractive option to prevent outbreaks of LPAIV in poultry.

  8. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  9. Rapid green synthesis of ZnO nanoparticles using a hydroelectric cell without an electrolyte

    Science.gov (United States)

    Shah, Jyoti; Kumar Kotnala, Ravinder

    2017-09-01

    In this study, zinc oxide (ZnO) nanoparticles were synthesized using a novel environmentally friendly hydroelectric cell without an electrolyte or external current source. The hydroelectric cell comprised a nanoporous Li substituted magnesium ferrite pellet in contact with two electrodes, with zinc as the anode and silver as an inert cathode. The surface unsaturated cations and oxygen vacancies in the nanoporous ferrite dissociated water molecules into hydronium and hydroxide ions when the hydroelectric cell was dipped into deionized water. Hydroxide ions migrated toward the zinc electrode to form zinc hydroxide and the hydronium ions were evolved as H2 gas at the silver electrode. The zinc hydroxide collected as anode mud was converted into ZnO nanoparticles by heating at 250 °C. Structural analysis using Raman spectroscopy indicated the good crystallinity of the ZnO nanoparticles according to the presence of a high intensity E2-(high) mode. The nanoparticle size distribution was 5-20 nm according to high resolution transmission electron microscopy. An indirect band gap of 2.75 eV was determined based on the Tauc plot, which indicated the existence of an interstitial cation level in ZnO. Near band edge and blue emissions were detected in photoluminescence spectral studies. The blue emissions obtained from the ZnO nanoparticles could potentially have applications in blue lasers and LEDs. The ZnO nanoparticles synthesized using this method had a high dielectric constant value of 5 at a frequency of 1 MHz, which could be useful for fabricating nano-oscillators. This facile, clean, and cost-effective method obtained a significant yield of 0.017 g for ZnO nanoparticles without applying an external current source.

  10. Rapid prototyping of microbial cell factories via genome-scale engineering.

    Science.gov (United States)

    Si, Tong; Xiao, Han; Zhao, Huimin

    2015-11-15

    Advances in reading, writing and editing genetic materials have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has greatly accelerated the cycles of design, build and test to engineer microbes for efficient synthesis of fuels, chemicals and drugs. In this review, we summarize the emerging technologies that have been applied, or are potentially useful for genome-scale engineering in microbial systems. We will focus on the development of high-throughput methodologies, which may accelerate the prototyping of microbial cell factories. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. AQP4e-Based Orthogonal Arrays Regulate Rapid Cell Volume Changes in Astrocytes.

    Science.gov (United States)

    Lisjak, Marjeta; Potokar, Maja; Rituper, Boštjan; Jorgačevski, Jernej; Zorec, Robert

    2017-11-01

    Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. It is predominantly expressed in astrocytes at the blood-brain and blood-liquor interfaces. Although several AQP4 isoforms have been identified in the mammalian brain, two, AQP4a (M1) and AQP4c (M23), have been confirmed to cluster into plasma membrane supramolecular structures, termed orthogonal arrays of particles (OAPs) and to enhance water transport through the plasma membrane. However, the role of the newly described water-conductive mammalian isoform AQP4e is unknown. Here, the dynamics of AQP4e aggregation into OAPs and its role in the regulation of astrocyte water homeostasis have been studied. Using super-resolution structured illumination, atomic force, and confocal microscopies, the results revealed that, in female rat astrocytes, AQP4e isoform colocalizes with OAPs, affecting its structural dynamics. In hypoosmotic conditions, which elicit cell edema, OAP formation was considerably enhanced by overexpressed AQP4e. Moreover, the kinetics of the cell swelling and of the regulatory volume decrease was faster in astrocytes overexpressing AQP4e compared with untransfected controls. Furthermore, the increase in maximal cell volume elicited by hypoosmotic stimulation was significantly smaller in AQP4e-overexpressing astrocytes. For the first time, this study demonstrates an active role of AQP4e in the regulation of OAP structural dynamics and in water homeostasis.SIGNIFICANCE STATEMENT Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. To date, only AQP4a and AQP4c isoforms have been confirmed to enhance water transport through plasmalemma and to cluster into orthogonal arrays of particles (OAPs). We here studied the dynamics, aggregation, and role in the regulation of astrocyte water homeostasis of the newly described water-conductive mammalian isoform AQP4e. Our main findings are as follows: brain edema

  12. Rapid changes in cell physiology as a result of acute thermal stress house sparrows, Passer domesticus.

    Science.gov (United States)

    Jimenez, Ana G; Williams, Joseph B

    2014-12-01

    Given that our climate is rapidly changing, Physiological Ecologists have the critical task of identifying characteristics of species that make them either resilient or susceptible to changes in their natural air temperature regime. Because climate change models suggest that heat events will become more common, and in some places more extreme, it is important to consider how extreme heat events might affect the physiology of a species. The implications of more frequent heat wave events for birds have only recently begun to be addressed, however, the impact of these events on the cellular physiology of a species is difficult to assess. We have developed a novel approach using dermal fibroblasts to explore how short-term thermal stress at the whole animal level might affect cellular rates of metabolism. House sparrows, Passer domesticus were separated into a "control group" and a "heat shocked" group, the latter acclimated to 43°C for 24h. We determined the plasticity of cellular thermal responses by assigning a "recovery group" that was heat shocked as above, but then returned to room temperature for 24h. Primary dermal fibroblasts were grown from skin of all treatment groups and the pectoralis muscle was collected. We found that glycolysis (ECAR) and oxygen consumption rates (OCR), measured using a Seahorse XF 96 analyzer, were significantly higher in the fibroblasts from the heat shocked group of House sparrows compared with their control counterparts. Additionally, muscle fiber diameters decreased and, in turn, Na(+)-K(+)-ATPase maximal activity in the muscle significantly increased in heat shocked sparrows compared with birds in the control group. All of these physiological alterations due to short-term heat exposure were reversible within 24h of recovery at room temperature. These results show that acute exposure to heat stress significantly alters the cellular physiology of sparrows, but that this species is plastic enough to recover from such a thermal

  13. Rapid and specific detection of cell-derived microvesicles using a magnetoresistive biochip.

    Science.gov (United States)

    Cherré, Solène; Fernandes, Elisabete; Germano, José; Dias, Tomás; Cardoso, Susana; Piedade, Moisés S; Rozlosnik, Noemi; Oliveira, Marta I; Freitas, Paulo P

    2017-03-13

    Microvesicles (MVs) are a promising source of diagnostic biomarkers which have gained a wide interest in the biomedical and biosensing field. They can be interpreted as a "fingerprint" of various diseases. Nonetheless, MVs implementation into clinical settings has been hampered by the lack of technologies to accurately characterize, detect and quantify them. Here, we report the specific sensing and quantification of MVs from endothelial cells using a portable magnetoresistive (MR) biochip platform, in less than one hour and within physiologically relevant concentrations (1 × 10(8) MVs per ml). MVs were isolated from both endothelial and epithelial cells undergoing apoptosis, and characterized by atomic force microscopy (AFM) and nanoparticle tracking analysis (NTA), which revealed similar MV sizes. Importantly, our results showed that the two distinct MV populations could be discriminated with the MR biochip platform, with over a 5-fold capture efficiency of endothelial MVs in comparison to the control (epithelial MVs). Also, unspecific binding of MVs to BSA was less than 1% of the specific signal. The detection strategy was based on a sandwich immunoassay, where MVs were labelled with magnetic nanoparticles (MNPs) functionalized with Annexin V and then captured by anti-CD31 antibodies previously immobilized on the surface of the sensor. Results suggest that this approach allows the detection of specific MVs from complex samples such as serum, and highlight the potential of this technology to become a suitable tool for MVs detection as a complementary method of diagnosis.

  14. Rapid detection of defects in fuel-cell electrodes using infrared reactive-flow-through technique

    Science.gov (United States)

    Das, Prodip K.; Weber, Adam Z.; Bender, Guido; Manak, Austin; Bittinat, Daniel; Herring, Andrew M.; Ulsh, Michael

    2014-09-01

    As fuel cells become more prominent, new manufacturing and production methods will need to be developed to deal efficiently and effectively with increased demand. One necessary component of this industrial growth is the accurate measurement of the variability in the manufacturing process. In this study, we present a diagnostic system that combines infrared thermography with a reactive-flow-through technique to detect catalyst-loading defects in fuel-cell gas-diffusion electrodes accurately with high spatial and temporal resolutions. Experimental results are compared with model predictions of thermal response with good agreement. Data analysis, operating-condition impacts, and detection limits are explored using both experiments and simulation. Overall, the results demonstrate the potential of this technique to measure defects on the millimeter length scale with temporal resolutions appropriate for use on a web-line. Thus we present the first development stage of a next-generation non-destructive diagnostic tool, which may be amenable to eventual use on roll-to-roll manufacturing lines.

  15. An easy, rapid method to isolate RPE cell protein from the mouse eye.

    Science.gov (United States)

    Wei, Hong; Xun, Zixian; Granado, Herta; Wu, Angela; Handa, James T

    2016-04-01

    The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis.

    Science.gov (United States)

    DuVall, Jacquelyn A; Cabaniss, Scott T; Angotti, Morgan L; Moore, John H; Abhyankar, Mayuresh; Shukla, Nishant; Mills, Daniel L; Kessel, Bryan G; Garner, Gavin T; Swami, Nathan S; Landers, James P

    2016-10-07

    Pathogen detection has traditionally been accomplished by utilizing methods such as cell culture, immunoassays, and nucleic acid amplification tests; however, these methods are not easily implemented in resource-limited settings because special equipment for detection and thermal cycling is often required. In this study, we present a magnetic bead aggregation assay coupled to an inexpensive microfluidic fabrication technique that allows for cell phone detection and analysis of a notable pathogen in less than one hour. Detection is achieved through the use of a custom-built system that allows for fluid flow control via centrifugal force, as well as manipulation of magnetic beads with an adjustable rotating magnetic field. Cell phone image capture and analysis is housed in a 3D-printed case with LED backlighting and a lid-mounted Android phone. A custom-written application (app.) is employed to interrogate images for the extent of aggregation present following loop-mediated isothermal amplification (LAMP) coupled to product-inhibited bead aggregation (PiBA) for detection of target sequences. Clostridium difficile is a pathogen of increasing interest due to its causative role in intestinal infections following antibiotic treatment, and was therefore chosen as the pathogen of interest in the present study to demonstrate the rapid, cost-effective, and sequence-specific detection capabilities of the microfluidic platform described herein.

  17. Bisphenol A induces a rapid activation of Erk1/2 through GPR30 in human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, S. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 (Japan); Institute of Urban Environment, Chinese Academy of Sciences, Xiamen (China); Terasaka, S. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 (Japan); Kiyama, R., E-mail: kiyama.r@aist.go.j [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 (Japan)

    2011-01-15

    Bisphenol A (BPA) has been considered as an endocrine disruptor due to its ability to interact with estrogen receptors (ERs). While G protein-coupled receptor 30 (GPR30) is a novel estrogen receptor, its role in BPA-induced activation of Erk1/2 remains unknown. Human breast cancer cell lines, MCF-7, MDA-MB-231 and SKBR3, were used as experimental models to discriminate between ERs-dependent, putative ERs-independent and/or GPR30-associated effects. BPA induced a rapid activation of Erk1/2 in both ER{alpha}/{beta}-positive and negative breast cancer cells, and this effect was not blocked with an ER antagonist, ICI 182,780. A small interfering RNA assay revealed that the expression of GPR30 was necessary for BPA-induced activation of Erk1/2 and transcriptional regulation of c-fos. In addition, BPA regulates the expression of c-fos likely through an AP1-mediated pathway. As a conclusion, GPR30 plays an important role in the BPA-induced activation of Erk1/2 in a manner distinguishable from that in ER{alpha}-mediated signaling. - We showed here that the mechanism by which BPA induces the activation of Erk1/2 is distinguishable from the mechanism of ER{alpha}-mediated signaling in human breast cancer cells.

  18. Extranodal NK/T-cell lymphoma, nasal type, manifesting as rapidly progressive dementia without any mass or enhancing brain lesion.

    Science.gov (United States)

    Shimatani, Yoshimitsu; Nakano, Yuta; Tsuyama, Naoko; Murayama, Shigeo; Oki, Ryosuke; Miyamoto, Ryosuke; Murakami, Nagahisa; Fujita, Koji; Watanabe, Syunsuke; Uehara, Hisanori; Abe, Takashi; Nodera, Hiroyuki; Kawarai, Toshitaka; Izumi, Yuishin; Kaji, Ryuji

    2016-10-01

    Among the many potential etiologies for rapidly progressive dementia (RPD), primary central nervous system extranodal NK/T-cell lymphoma, nasal-type (ENKL) is a rare entity. We present the first reported case of autopsy-proven RPD due to ENKL without any mass or enhancing lesion of the brain. A 54-year-old immunocompetent man presented with RPD, myoclonus and ataxia. The mini-mental state examination (MMSE) score was 22/30. His brain MRI revealed progressive brain atrophy without gadolinium enhancement or mass lesion. Five months after the initial evaluation, cognitive impairment further worsened with an MMSE score of 3/30. At the advanced stage, lumbar MRI showed swollen cauda equina with gadolinium enhancement. The number of Epstein-Barr virus (EBV) DNA in cerebrospinal fluid had gradually increased. Twelve months after onset, the patient died of respiratory failure. Pathological findings revealed that lymphoma cells had diffusely invaded the meninges, parenchyma of the brain, spinal cord and cauda equina. Cells were positive for CD3, CD56 and EBV-encoded small RNAs and negative for CD20. No evidence of malignancy was identified in the visceral organs. This report indicates that ENKL should be recognized as one of the rare causes of RPD. Early testing for EBV-DNA in cerebrospinal fluid and imaging of cauda equina would be useful diagnostic tools. © 2016 Japanese Society of Neuropathology.

  19. Candida albicans and Candida parapsilosis rapidly up-regulate galectin-3 secretion by human gingival epithelial cells.

    Science.gov (United States)

    Tamai, Riyoko; Kiyoura, Yusuke

    2014-02-01

    Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.

  20. A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

    NARCIS (Netherlands)

    Schalk, Johanna A. C.; de Vries, Claudette G. J. C. A.; Orzechowski, Tom J. H.; Rots, Marianne G.

    2007-01-01

    The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by

  1. An enzyme-activatable probe with a self-immolative linker for rapid and sensitive alkaline phosphatase detection and cell imaging through a cascade reaction.

    Science.gov (United States)

    Zhang, Hongmei; Xu, Chenglong; Liu, Jie; Li, Xiaohong; Guo, Lin; Li, Xinming

    2015-04-25

    We report the design and synthesis of a novel probe (1) for ALP assay by incorporating a self-immolative linker between a phosphate moiety and resorufin. Because of its good biocompatibility and rapid cell internalization, this probe also exhibited great potential for real-time monitoring of endogenous phosphatase activity in living cells.

  2. Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell

    Directory of Open Access Journals (Sweden)

    Ji Yeon Hwang

    2016-11-01

    Full Text Available Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule or muc1 (mucin1 expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon. The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1 or BHQ2 (black hole quencher2. In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.

  3. Cobalamin C Deficiency Shows a Rapidly Progressing Maculopathy With Severe Photoreceptor and Ganglion Cell Loss

    Science.gov (United States)

    Bonafede, Lucas; Ficicioglu, Can H.; Serrano, Leona; Han, Grace; Morgan, Jessica I. W.; Mills, Monte D.; Forbes, Brian J.; Davidson, Stefanie L.; Binenbaum, Gil; Kaplan, Paige B.; Nichols, Charles W.; Verloo, Patrick; Leroy, Bart P.; Maguire, Albert M.; Aleman, Tomas S.

    2015-01-01

    Purpose To describe in detail the retinal structure and function of a group of patients with cobalamin C (cblC) disease. Methods Patients (n = 11, age 4 months to 15 years) with cblC disease (9/11, early onset) diagnosed by newborn screening underwent complete ophthalmic examinations, fundus photography, near-infrared reflectance imaging, and spectral-domain optical coherence tomography (SD-OCT). Electroretinograms (ERGs) were performed in a subset of patients. Results Patients carried homozygous or compound heterozygote mutations in the methylmalonic aciduria and homocystinuria type C (MMACHC) gene. Late-onset patients had a normal exam. All early-onset patients showed a maculopathy; older subjects had a retina-wide degeneration (n = 4; >7 years of age). In general, retinal changes were first observed before 1 year of age and progressed within months to a well-established maculopathy. Pseudocolobomas were documented in three patients. Measurable visual acuities ranged from 20/200 to 20/540. Nystagmus was present in 8/11 patients; 5/6 patients had normal ERGs; 1/6 had reduced rod-mediated responses. Spectral-domain OCT showed macular thinning, with severe ganglion cell layer (GCL) and outer nuclear layer (ONL) loss. Inner retinal thickening was observed in areas of total GCL/ONL loss. A normal lamination pattern in the peripapillary nasal retina was often seen despite severe central and/or retina-wide disease. Conclusions Patients with early-onset cblC and MMACHC mutations showed an early-onset, unusually fast-progressing maculopathy with severe central ONL and GCL loss. An abnormally thickened inner retina supports a remodeling response to both photoreceptor and ganglion cell degeneration and/or an interference with normal development in early-onset cblC. PMID:26658511

  4. A paper-based microbial fuel cell array for rapid and high-throughput screening of electricity-producing bacteria.

    Science.gov (United States)

    Choi, Gihoon; Hassett, Daniel J; Choi, Seokheun

    2015-06-21

    There is a large global effort to improve microbial fuel cell (MFC) techniques and advance their translational potential toward practical, real-world applications. Significant boosts in MFC performance can be achieved with the development of new techniques in synthetic biology that can regulate microbial metabolic pathways or control their gene expression. For these new directions, a high-throughput and rapid screening tool for microbial biopower production is needed. In this work, a 48-well, paper-based sensing platform was developed for the high-throughput and rapid characterization of the electricity-producing capability of microbes. 48 spatially distinct wells of a sensor array were prepared by patterning 48 hydrophilic reservoirs on paper with hydrophobic wax boundaries. This paper-based platform exploited the ability of paper to quickly wick fluid and promoted bacterial attachment to the anode pads, resulting in instant current generation upon loading of the bacterial inoculum. We validated the utility of our MFC array by studying how strategic genetic modifications impacted the electrochemical activity of various Pseudomonas aeruginosa mutant strains. Within just 20 minutes, we successfully determined the electricity generation capacity of eight isogenic mutants of P. aeruginosa. These efforts demonstrate that our MFC array displays highly comparable performance characteristics and identifies genes in P. aeruginosa that can trigger a higher power density.

  5. Bridging the transatlantic divide in privacy

    Directory of Open Access Journals (Sweden)

    Paula Kift

    2013-08-01

    Full Text Available In the context of the US National Security Agency surveillance scandal, the transatlantic privacy divide has come back to the fore. In the United States, the right to privacy is primarily understood as a right to physical privacy, thus the protection from unwarranted government searches and seizures. In Germany on the other hand, it is also understood as a right to spiritual privacy, thus the right of citizens to develop into autonomous moral agents. The following article will discuss the different constitutional assumptions that underlie American and German attitudes towards privacy, namely privacy as an aspect of liberty or as an aspect of dignity. As data flows defy jurisdictional boundaries, however, policymakers across the Atlantic are faced with a conundrum: how can German and American privacy cultures be reconciled?

  6. Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner.

    Directory of Open Access Journals (Sweden)

    John Meshki

    Full Text Available U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R. Substance P (SP, the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.

  7. Microbial fuel cell biosensor for rapid assessment of assimilable organic carbon under marine conditions.

    Science.gov (United States)

    Quek, Soon Bee; Cheng, Liang; Cord-Ruwisch, Ralf

    2015-06-15

    The development of an assimilable organic carbon (AOC) detecting marine microbial fuel cell (MFC) biosensor inoculated with microorganisms from marine sediment was successful within 36 days. This established marine MFC was tested as an AOC biosensor and reproducible microbiologically produced electrical signals in response to defined acetate concentration were achieved. The dependency of the biosensor sensitivity on the potential of the electron-accepting electrode (anode) was investigated. A linear correlation (R(2) > 0.98) between electrochemical signals (change in anodic potential and peak current) and acetate concentration ranging from 0 to 150 μM (0-3600 μg/L of AOC) was achieved. However, the present biosensor indicated a different-linear relation at somewhat elevated acetate concentration ranging from 150 to 450 μM (3600-10,800 μg/L of AOC). This high concentration of acetate addition could be measured by coulombic measurement (cumulative charges) with a linear correlation. For the acetate concentration detected in this study, the sensor recovery time could be controlled within 100 min. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Novel photosensitizers trigger rapid death of malignant human cells and rodent tumor transplants via lipid photodamage and membrane permeabilization.

    Directory of Open Access Journals (Sweden)

    Mikhail M Moisenovich

    Full Text Available BACKGROUND: Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. METHODOLOGY/PRINCIPAL FINDINGS: Our novel derivatives of chlorin e(6, that is, its amide (compound 2 and boronated amide (compound 5 evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e(6 against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3-4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e(6 in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. CONCLUSIONS/SIGNIFICANCE: The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are

  9. Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells

    Science.gov (United States)

    Kalinkovich, Alexander; Itkin, Tomer; Ludin, Aya; Kao, Wei-Ming; Battista, Michela; Tesio, Melania; Kollet, Orit; Cohen, Neta Netzer; Margalit, Raanan; Buss, Eike C.; Baleux, Francoise; Oishi, Shinya; Fujii, Nobutaka; Larochelle, Andre; Dunbar, Cynthia E.; Broxmeyer, Hal E.; Frenette, Paul S.; Lapidot, Tsvee

    2014-01-01

    Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine SDF-1 to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady-state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, while β2-adrenergic antagonist inhibited both steady-state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes. PMID:21494253

  10. Metformin: a case of divide and conquer

    Science.gov (United States)

    2013-01-01

    Metformin is a widely prescribed anti-diabetic drug and its use is associated with lower cancer incidence. The mechanisms by which metformin attenuates tumorigenesis are not clearly understood. In a paper published in Proceedings of the National Academy of Sciences of the United States of America, Hirsch and colleagues show that metformin interferes with a signaling pathway, mediated by the transcription factor NF-κB, which drives cell transformation and is required for the maintenance of cancer stem cells. PMID:23510106

  11. Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

    Science.gov (United States)

    De Sousa, Paul A; Steeg, Rachel; Wachter, Elisabeth; Bruce, Kevin; King, Jason; Hoeve, Marieke; Khadun, Shalinee; McConnachie, George; Holder, Julie; Kurtz, Andreas; Seltmann, Stefanie; Dewender, Johannes; Reimann, Sascha; Stacey, Glyn; O'Shea, Orla; Chapman, Charlotte; Healy, Lyn; Zimmermann, Heiko; Bolton, Bryan; Rawat, Trisha; Atkin, Isobel; Veiga, Anna; Kuebler, Bernd; Serano, Blanca Miranda; Saric, Tomo; Hescheler, Jürgen; Brüstle, Oliver; Peitz, Michael; Thiele, Cornelia; Geijsen, Niels; Holst, Bjørn; Clausen, Christian; Lako, Majlinda; Armstrong, Lyle; Gupta, Shailesh K; Kvist, Alexander J; Hicks, Ryan; Jonebring, Anna; Brolén, Gabriella; Ebneth, Andreas; Cabrera-Socorro, Alfredo; Foerch, Patrik; Geraerts, Martine; Stummann, Tina C; Harmon, Shawn; George, Carol; Streeter, Ian; Clarke, Laura; Parkinson, Helen; Harrison, Peter W; Faulconbridge, Adam; Cherubin, Luca; Burdett, Tony; Trigueros, Cesar; Patel, Minal J; Lucas, Christa; Hardy, Barry; Predan, Rok; Dokler, Joh; Brajnik, Maja; Keminer, Oliver; Pless, Ole; Gribbon, Philip; Claussen, Carsten; Ringwald, Annette; Kreisel, Beate; Courtney, Aidan; Allsopp, Timothy E

    2017-04-01

    A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Microfabricated Renewable Beads-Trapping/Releasing Flow Cell for Rapid Antigen-Antibody Reaction in Chemiluminescent Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Zhifeng; Shao, Guocheng; Wang, Jun; Lu, Donglai; Wang, Wanjun; Lin, Yuehe

    2011-04-01

    A filter pillar-array microstructure was coupled with a pneumatic micro-valve to fabricate a reusable miniaturized beads-trapping/releasing flow cell, in which trapping and releasing beads can be conveniently realized by switching the micro-valve. This miniaturized device was suitable to construct automatic fluidic system for “renewable surface analysis”. The renewable surface strategy based on pneumatic micro-valve enabled capture of beads in beads chamber prior to each assay, and release of the used beads after the assay. Chemiluminescent competitive immunoassay of 3,5,6-trichloropyridinol (TCP) was performed as a model to demonstrate the application potential of this reusable miniaturized flow cell. The whole fluidic assay process including beads trapping, immuno-binding, beads washing, beads releasing and signal collection could be completed in 10 min. Immunoassay of TCP using this miniaturized device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device had been successfully used for detection of TCP spiked in rat serum with average recovery of 97%. This investigation provides a rapid, sensitive, reusable, low-cost and automatic miniaturized device for solid-phase biochemical analysis for various purposes.

  13. Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones.

    Science.gov (United States)

    Dambrot, Cheryl; Buermans, Henk P J; Varga, Eszter; Kosmidis, Georgios; Langenberg, Karin; Casini, Simona; Elliott, David A; Dinnyes, Andras; Atsma, Douwe E; Mummery, Christine L; Braam, Stefan R; Davis, Richard P

    2014-10-01

    Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Rapid fabrication of mesoporous TiO2 thin films by pulsed fibre laser for dye sensitized solar cells

    Science.gov (United States)

    Hadi, Aseel; Alhabradi, Mansour; Chen, Qian; Liu, Hong; Guo, Wei; Curioni, Michele; Cernik, Robert; Liu, Zhu

    2018-01-01

    In this paper we demonstrate for the first time that a fibre laser with a wavelength of 1070 nm and a pulse width of milliseconds can be applied to generate mesoporous nanocrystalline (nc) TiO2 thin films on ITO coated glass in ambient atmosphere, by complete vaporisation of organic binder and inter-connection of TiO2 nanoparticles, without thermally damaging the ITO layer and the glass substrate. The fabrication of the mesoporous TiO2 thin films was achieved by stationary laser beam irradiation of 1 min. The dye sensitized solar cell (DSSC) with the laser-sintered TiO2 photoanode reached higher power conversion efficiency (PCE) of 3.20% for the TiO2 film thickness of 6 μm compared with 2.99% for the furnace-sintered. Electrochemical impedance spectroscopy studies revealed that the laser sintering under the optimised condition effectively decreased charge transfer resistance and increased electron lifetime of the TiO2 thin films. The use of the fibre laser with over 40% wall-plug efficiency offers an economically-feasible, industrial viable solution to the major challenge of rapid fabrication of large scale, mass production of mesoporous metal oxide thin film based solar energy systems, potentially for perovskite and monolithic tandem solar cells, in the future.

  15. Rapid generation of hydrogen peroxide contributes to the complex cell death induction by the angucycline antibiotic landomycin E.

    Science.gov (United States)

    Panchuk, Rostyslav R; Lehka, Lilya V; Terenzi, Alessio; Matselyukh, Bohdan P; Rohr, Jürgen; Jha, Amit K; Downey, Theresa; Kril, Iryna J; Herbacek, Irene; van Schoonhoven, Sushilla; Heffeter, Petra; Stoika, Rostyslav S; Berger, Walter

    2017-05-01

    Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH(•) effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main

  16. The Gender Digital Divide in Developing Countries

    Directory of Open Access Journals (Sweden)

    Amy Antonio

    2014-10-01

    Full Text Available Empirical studies clearly show that women in the developing world have significantly lower technology participation rates than men; a result of entrenched socio-cultural attitudes about the role of women in society. However, as studies are beginning to show, when those women are able to engage with Internet technology, a wide range of personal, family and community benefits become possible. The key to these benefits is on-line education, the access to which sets up a positive feedback loop. This review gives an overview of the digital divide, before focusing specifically on the challenges women in developing countries face in accessing the Internet. Current gender disparities in Internet use will be outlined and the barriers that potentially hinder women’s access and participation in the online world will be considered. We will then look at the potential opportunities for women’s participation in a global digital society along with a consideration of current initiatives that have been developed to mitigate gender inequity in developing countries. We will also consider a promising avenue for future research.

  17. The Digital Divide and urban older adults.

    Science.gov (United States)

    Cresci, M Kay; Yarandi, Hossein N; Morrell, Roger W

    2010-01-01

    Computers and the Internet offer older adults opportunities and resources for independent living. However, many urban older adults do not use computers. This study examined the demographic, health, and social activities of urban older adults to determine variables that might predict the use and nonuse of computers in this population. A secondary data analysis was performed using the 2001 Detroit City-Wide Needs Assessment of Older Adults (n = 1410) data set. Logistic regression was used to explore potential differences in predictor variables between computer users and nonusers. Overall, computer users were younger (27%), had a higher level of education, were more likely to be employed, had an annual income greater than $20,000, and were healthier and more active than nonusers. They also were more likely to have memberships in community organizations and do volunteer work. Preferred computer activities included conducting Internet searches, playing games, writing, and communicating with family members and friends. The results suggest significant differences in demographic and health-related characteristics between computer users and nonusers among urban older adults. Although about a quarter of participants in this study used computers, the Digital Divide continues to exist in urban settings for scores of others.

  18. Bridging the divide between science and journalism.

    Science.gov (United States)

    Van Eperen, Laura; Marincola, Francesco M; Strohm, Jennifer

    2010-03-10

    There are countless reasons nearly every scientist should learn how to communicate effectively with the media, including increased understanding of critical research findings to attract or sustain funding and build new professional partnerships that will further propel forward research. But where do scientists begin? Bridging the Divide between Science and Journalism offers practical tips for any scientist looking to work with the media.Given the traditional and internet-based sources for medical research and healthcare-related news now available, it is imperative that scientists know how to communicate their latest findings through the appropriate channels. The credible media channels are managed by working journalists, so learning how to package vast, technical research in a form that is appetizing and "bite-sized" in order to get their attention, is an art. Reducing years of research into a headline can be extremely difficult and certainly doesn't come naturally to every scientist, so this article provides suggestions on how to work with the media to communicate your findings.

  19. Bridging the Divide between Science and Journalism

    Directory of Open Access Journals (Sweden)

    Marincola Francesco M

    2010-03-01

    Full Text Available Abstract There are countless reasons nearly every scientist should learn how to communicate effectively with the media, including increased understanding of critical research findings to attract or sustain funding and build new professional partnerships that will further propel forward research. But where do scientists begin? Bridging the Divide between Science and Journalism offers practical tips for any scientist looking to work with the media. Given the traditional and internet-based sources for medical research and healthcare-related news now available, it is imperative that scientists know how to communicate their latest findings through the appropriate channels. The credible media channels are managed by working journalists, so learning how to package vast, technical research in a form that is appetizing and "bite-sized" in order to get their attention, is an art. Reducing years of research into a headline can be extremely difficult and certainly doesn't come naturally to every scientist, so this article provides suggestions on how to work with the media to communicate your findings.

  20. The private-public law divide

    DEFF Research Database (Denmark)

    Svendsen, Idamarie Leth; Kronborg, Annette; Gyldenløve Jeppesen-De Boer, Christina

    2013-01-01

    In a Scandinavian perspective, the family and the individual have changed places during the 20th Century. Today, the law takes its starting point in the individual - not in the family. A consequence of this development is that it is no longer legally possible to relate the good family to a partic......In a Scandinavian perspective, the family and the individual have changed places during the 20th Century. Today, the law takes its starting point in the individual - not in the family. A consequence of this development is that it is no longer legally possible to relate the good family...... to a particular societal institution. Marriage as an institution has been individualized and the goodness of the family has to relate to the well-being of the individuals instead. This article shows that within this historical development the private-public law divide has not been seriously challenged...... that the historical development neccessitates a re-thinking of the distinction between child welfare law and family law. It shows how the distinction is nationally and institutionally embedded. Further, that the distinction has only been superficially adressed by the CRC Committee....

  1. Synthetic virus-like particles target dendritic cell lipid rafts for rapid endocytosis primarily but not exclusively by macropinocytosis.

    Directory of Open Access Journals (Sweden)

    Rajni Sharma

    Full Text Available DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20-30 nm virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1(+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.

  2. Thymic irradiation inhibits the rapid recovery of TH1 but not TH2-like functions of CD4+ T cells after total lymphoid irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bass, H.; Adkins, B.; Strober, S. (Department of Medicine, Stanford University School of Medicine, CA (United States))

    1991-10-15

    Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.

  3. The Digital Divide in Romania – A Statistical Analysis

    Directory of Open Access Journals (Sweden)

    Daniela BORISOV

    2012-06-01

    Full Text Available The digital divide is a subject of major importance in the current economic circumstances in which Information and Communication Technologies (ICT are seen as a significant determinant of increasing the domestic competitiveness and contribute to better life quality. Latest international reports regarding various aspects of ICT usage in modern society reveal a decrease of overall digital disparity towards the average trends of the worldwide ITC’s sector – this relates to latest advances of mobile and computer penetration rates, both for personal use and for households/ business. In Romania, the low starting point in the development of economy and society in the ICT direction was, in some extent, compensated by the rapid annual growth of the last decade. Even with these dynamic developments, the statistical data still indicate poor positions in European Union hierarchy; in this respect, the prospects of a rapid recovery of the low performance of the Romanian ICT endowment and usage and the issue continue to be regarded as a challenge for progress in economic and societal terms. The paper presents several methods for assessing the current state of ICT related aspects in terms of Internet usage based on the latest data provided by international databases. The current position of Romanian economy is judged according to several economy using statistical methods based on variability measurements: the descriptive statistics indicators, static measures of disparities and distance metrics.

  4. Physician executives straddle the digital divide.

    Science.gov (United States)

    Coile, R C

    2001-01-01

    e-Health is here to stay and experts predict that the Internet will become the hub of health care. Rapid advancements in biotechnology and medical research, increasingly curious patients who surf the Internet for medical information, and pressures from managed care companies to contain costs and speed treatments are the central components driving e-health. Despite physician reluctance to embrace the e-revolution, many hospitals and medical groups are employing the Internet and information technology to improve their customer interface, as well as to reduce business costs. This article offers seven e-strategies for health care performance improvement: (1) Supply chain management; (2) e-transactions; (3) care management; (4) improving quality; (5) boosting revenues; (6) outsourcing; and (7) provider networks (Intranets). By helping to incorporate these key e-solutions, physician executives can position their organizations for success in the new millennium.

  5. Plasticity of intestinal epithelial cells in regeneration and cancer

    NARCIS (Netherlands)

    Tetteh, Paul W.

    2015-01-01

    Cellular plasticity refers to the ability of a cell to change its fate or identity in response to external or intrinsic factors. Regeneration of the intestinal epithelium after injury is driven mainly by plasticity of crypt stem cells that can rapidly divide to replace all the lost cells. Stem cell

  6. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

    Science.gov (United States)

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan

    2016-09-01

    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  7. Rapid reduction of MDCK cell cholesterol by methyl-beta-cyclodextrin alters steady state transepithelial electrical resistance.

    Science.gov (United States)

    Francis, S A; Kelly, J M; McCormack, J; Rogers, R A; Lai, J; Schneeberger, E E; Lynch, R D

    1999-07-01

    The role of plasma membrane lipids in regulating the passage of ions and other solutes through the paracellular pathway remains controversial. In this study we explore the contribution of cholesterol (CH) in maintaining the barrier function of an epithelial cell line using the CH-solubilizing agent methyl beta-cyclodextrin (MBCD) to stimulate CH efflux. Inclusion of 20 mM MBCD in both apical and basolateral media reduced CH levels by 70-80% with no significant effect on cell viability. Most of that decrease occurred during the first 30 min of incubation. Recovery of CH content to initial values was nearly complete 22 h after removal of MBCD. Within 30 min of adding MBCD to the culture medium, transepithelial electrical resistance (TER) increased, reaching maximum values 30-40% above controls. This early rise in TER occurred when MBCD was added to either side of the monolayer. The later rapid decline in TER was observed only when MBCD bathed the basolateral surface from which, coincidentally, CH efflux was most rapid. Freeze fracture replicas and transmission electron microscopy of monolayers exposed to MBCD for only 30 min revealed no increase in either the average tight junction (TJ) strand number or the dimensions of the lateral intercellular space. There was a statistically significant increase in the number of TJ particles associated with the E fracture face at this time. This raises the interesting possibility that during CH efflux there is a change in the interaction between TJ particles and underlying cytoskeletal elements. There was no change in staining for occludin and ZO-1. After exposing the basolateral surface to MBCD for 2 h, TER fell below control levels. The accompanying increase in mannitol flux suggests strongly that the decrease in TER resulted from an increase in the permeability of the paracellular and not the transcellular pathway. A decrease in immuno-staining for occludin and ZO-1 at TJs, a striking accumulation of actin at tri

  8. Rapid engraftment without significant graft-versus-host disease after allogeneic transplantation of CD34+ selected cells from peripheral blood.

    Science.gov (United States)

    Urbano-Ispizua, A; Rozman, C; Martínez, C; Marín, P; Briones, J; Rovira, M; Féliz, P; Viguria, M C; Merino, A; Sierra, J; Mazzara, R; Carreras, E; Montserrat, E

    1997-06-01

    have relapsed, and one of them is again in hematologic and cytogenetic remission after infusion of the donor lymphocytes. Two patients died in remission: one on day +109 of pulmonary aspergillosis and the other on day +251 of metastasic relapse of a previous breast cancer. Sixteen of the 20 patients are alive in remission after a median follow-up of 7.5 months (range, 2 to 22). In conclusion, despite the small number of patients and limited follow-up, it appears that this method allows a high CD34+ cell recovery from G-CSF mobilized PBPC and is associated with rapid engraftment without significant GVHD, and with low transplant related mortality.

  9. In vitro phospholipid biosynthesis for growing and dividing minimal cells

    NARCIS (Netherlands)

    Scott, A.D.D.

    2016-01-01

    Natural life is extraordinarily complex, which by definition means that it has many interconnected and functioning parts. The goal of synthetic biology is to engineer living systems, though due to their very complexity they remain recalcitrant to engineering. What if it were possible to reduce the

  10. Mechanisms of Mutation in Non-Dividing Cells

    Science.gov (United States)

    2003-05-01

    strains and plasmids formation in the TGV system (14). SMR5201, a transformant Strain or Reference carrying ampRC+ replacing attX (confirmed by PCR as de...6 0.895 1.4 structed using the TGV (transgenic vector) system for integrat- 7 1.69 1.4 ing linear DNA cassettes into the E. coli chromosome (14). E. I...negative bacteria: global trends and clinical impact. Clin. Infect. Dis. 15:824- berg. 2001. The TGV transgenic vectors for single-copy gene expression

  11. Critical role of perforin-dependent CD8+ T cell immunity for rapid protective vaccination in a murine model for human smallpox.

    Directory of Open Access Journals (Sweden)

    Melanie Kremer

    Full Text Available Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination.

  12. Critical role of perforin-dependent CD8+ T cell immunity for rapid protective vaccination in a murine model for human smallpox.

    Science.gov (United States)

    Kremer, Melanie; Suezer, Yasemin; Volz, Asisa; Frenz, Theresa; Majzoub, Monir; Hanschmann, Kay-Martin; Lehmann, Michael H; Kalinke, Ulrich; Sutter, Gerd

    2012-01-01

    Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s) of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA) or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination.

  13. Cytokine-induced proapoptotic gene expression in insulin-producing cells is related to rapid, sustained, and nonoscillatory nuclear factor-kappaB activation

    DEFF Research Database (Denmark)

    Ortis, Fernanda; Cardozo, Alessandra K; Crispim, Daisy

    2006-01-01

    Cytokines, such as IL-1beta and TNF-alpha, contribute to pancreatic beta-cell death in type 1 diabetes mellitus. The transcription factor nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced beta-cell apoptosis. Paradoxically, NF-kappaB has mostly antiapoptotic effects in other cell types......-kappaB activation in insulin-producing cells is more rapid, marked, and sustained than in fibroblasts, which correlates with a more pronounced activation of downstream genes and a proapoptotic outcome....

  14. "Enlivening and - Dividing": An Aporia of Illumination

    Directory of Open Access Journals (Sweden)

    Hans Christian Hönes

    2015-08-01

    Full Text Available Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Verdana;} In 1798, Karl August Böttiger paid a nocturnal visit to the Gallery of Antiques in Dresden, illuminating the statues with a torch. At first glance, this seems to be yet another example of a popular practice for visiting galleries c.1800. Illuminating the sculptures by torchlight was a popular means of enlivening the objects, set in motion by the light flickering on their surfaces. The collections were thus meant to become a place where cold, white stone comes to life, and where the beholder becomes part of a revived antiquity.This was precisely what Böttiger intended, too. But to him, the effect of the torchlight appeared to be, as he wrote, “enlivening and – dividing!” The torchlight highlighted not only the beauty of the sculptures but also their modern restorations. Böttiger apparently failed to experience the living presence of the antique celebrated by many of his contemporaries (e.g. Goethe, Moritz.This essay focuses on the consequence of such a perception of sculptures as historically multi-layered objects. Böttiger’s experience resulted in a problematic situation. In trying to view the sculptures as contemporaries, he hoped to become ancient himself. But this operation failed in the moment when the sculptures themselves appeared to be anachronistic, impure palimpsests. In consequence, galleries may not only be the place were art history as chronological Stilgeschichte was born. They may also be the site where this perception changed into the experience of a more chaotic shape of time.

  15. A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthe.

    Science.gov (United States)

    Gan, Rui; Jewett, Michael C

    2014-05-01

    Cell-free protein synthesis (CFPS) provides a valuable platform for understanding, using, and expanding the capabilities of the translation apparatus. For example, high-throughput CFPS is helping to address the increasing discrepancy between genome sequence data and their translation products. Here, we report the development of a combined cell-free transcription-translation (Tx/Tl) system from Saccharomyces cerevisiae that is suitable for such efforts. First, we show the ability to enable translation initiation in a cap-independent manner. The performance of various genetic elements was assessed, including 5'-UTR, 3'-UTR, and length of poly(A) tail. A specific vector harboring the 5'-UTR fragment of the Ω sequence from the tobacco mosaic virus and a poly(A) tail of 50 nucleotides led to optimal performance. Second, we developed a simple, two-step polymerase chain reaction (PCR) method for high-throughput production of linear templates for yeast CFPS. This procedure allows all functional elements needed for Tx/Tl to be added to an open-reading frame directly by overlap extension PCR. Our two-step PCR method was successfully applied to three reporter proteins: luciferase, green fluorescence protein, and chloramphenicol acetyl transferase, yielding 7 to 12.5 μg mL-1 active protein after 1.5-h batch reactions. Surprisingly, the linear templates outperformed plasmid DNA by up to 60%. Hence, the presented CFPS method has the potential to rapidly prepare tens to thousands of DNA templates without time-consuming cloning work. Further, it holds promise for fast and convenient optimization of expression constructs, study of internal ribosome entry site, and production of protein libraries for genome-scale studies. See accompanying commentary by Russ and Dueber DOI: 10.1002/biot.201400071. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Small-Wins Perspective: Educators Have a Role to Play in Narrowing the Generation Divide

    Science.gov (United States)

    Nichols, Sharon L.; Good, Thomas L.

    2005-01-01

    Adults widely misunderstand youth. Although this is not a new phenomenon--adults have been baffled by teenagers for centuries--a rapidly changing media environment coupled with the increasing demands placed on the youth have fueled the growing generation divide in unprecedented ways. Indeed the modern, fast-paced, multi-tasked, highly mobile,…

  17. Rapid thermal annealing of sputter-deposited ZnO:Al films for microcrystalline Si thin-film solar cells

    Directory of Open Access Journals (Sweden)

    Hanajiri T.

    2012-06-01

    Full Text Available Rapid thermal annealing of sputter-deposited ZnO and Al-doped ZnO (AZO films with and without an amorphous silicon (a-Si capping layer was investigated using a radio-frequency (rf argon thermal plasma jet of argon at atmospheric pressure. The resistivity of bare ZnO films on glass decreased from 108 to 104–105 Ω cm at maximum surface temperatures Tmaxs above 650 °C, whereas the resistivity increased from 10-4 to 10-3–10-2Ω cm for bare AZO films. On the other hand, the resistivity of AZO films with a 30-nm-thick a-Si capping layer remained below 10-4Ω cm, even after TPJ annealing at a Tmax of 825 °C. The film crystallization of both AZO and a-Si layers was promoted without the formation of an intermixing layer. Additionally, the crystallization of phosphorous- and boron-doped a-Si layers at the sample surface was promoted, compared to that of intrinsic a-Si under the identical plasma annealing conditions. The TPJ annealing of n+-a-Si/textured AZO was applied for single junction n-i-p microcrystalline Si thin-film solar cells.

  18. Membrane stress is coupled to a rapid translational control of gene expression in chlorpromazine-treated cells.

    Science.gov (United States)

    De Filippi, Loic; Fournier, Margot; Cameroni, Elisabetta; Linder, Patrick; De Virgilio, Claudio; Foti, Michelangelo; Deloche, Olivier

    2007-09-01

    Chlorpromazine (CPZ) is a small permeable cationic amphiphilic molecule that inserts into membrane bilayers and binds to anionic lipids such as poly-phosphoinositides (PIs). Since PIs play important roles in many cellular processes, including signaling and membrane trafficking pathways, it has been proposed that CPZ affects cellular growth functions by preventing the recruitment of proteins with specific PI-binding domains. In this study, we have investigated the biological effects of CPZ in the yeast Saccharomyces cerevisiae. We screened a collection of approximately 4,800 gene knockout mutants, and found that mutants defective in membrane trafficking between the late-Golgi and endosomal compartments are highly sensitive to CPZ. Microscopy and transport analyses revealed that CPZ affects membrane structure of organelles, blocks membrane transport and activates the unfolded protein response (UPR). In addition, CPZ-treatment induces phosphorylation of the translation initiation factor (eIF2alpha), which reduces the general rate of protein synthesis and stimulates the production of Gcn4p, a major transcription factor that is activated in response to environmental stresses. Altogether, our results reveal that membrane stress within the cells rapidly activates an important gene expression program, which is followed by a general inhibition of protein synthesis. Remarkably, the increase of phosphorylated eIF2alpha and protein synthesis inhibition were also detected in CPZ-treated NIH-3T3 fibroblasts, suggesting the existence of a conserved mechanism of translational regulation that operates during a membrane stress.

  19. Rapid atmospheric pressure plasma jet processed reduced graphene oxide counter electrodes for dye-sensitized solar cells.

    Science.gov (United States)

    Liu, Hsiao-Wei; Liang, Sheng-Ping; Wu, Ting-Jui; Chang, Haoming; Kao, Peng-Kai; Hsu, Cheng-Che; Chen, Jian-Zhang; Chou, Pi-Tai; Cheng, I-Chun

    2014-09-10

    In this work, we present the use of reduced graphene oxide (rGO) as the counter electrode materials in dye-sensitized solar cells (DSSCs). rGO was first deposited on a fluorine-doped tin oxide glass substrate by screen-printing, followed by post-treatment to remove excessive organic additives. We investigated the effect of atmospheric pressure plasma jet (APPJ) treatment on the DSSC performance. A power conversion efficiency of 5.19% was reached when DSSCs with an rGO counter electrode were treated by APPJs in the ambient air for a few seconds. For comparison, it requires a conventional calcination process at 400 °C for 15 min to obtain comparable efficiency. Scanning electron micrographs show that the APPJ treatment modifies the rGO structure, which may reduce its conductivity in part but simultaneously greatly enhances its catalytic activity. Combined with the rapid removal of organic additives by the highly reactive APPJ, DSSCs with APPJ-treated rGO counter electrode show comparable efficiencies to furnace-calcined rGO counter electrodes with greatly reduced process time. This ultrashort process time renders an estimated energy consumption per unit area of 1.1 kJ/cm(2), which is only one-third of that consumed in a conventional furnace calcination process. This new methodology thus saves energy, cost, and time, which is greatly beneficial to future mass production.

  20. Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.

    Directory of Open Access Journals (Sweden)

    Jens Durruthy-Durruthy

    Full Text Available Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP. Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

  1. Rapid procedure for coupling of protein antigens to red cells to be used in plaque assays by prewashing in chromium chloride

    NARCIS (Netherlands)

    Savelkoul, H.F.J.; Greeve, A.A.M.; Rijkers, G.T.; Marwitz, P.A.; Benner, R.

    1988-01-01

    A rapid and efficient procedure is described for the coupling of proteins (protein A, provalbumin, albumin and chicken gamma globulin) to sheep red blood cells (SRBC) to be used in antigen-specific or protein A plaque assays. This modification of the original procedure has three distinct features:

  2. Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Serrano Oscar K

    2008-10-01

    Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

  3. Rapid dimerization of quercetin through an oxidative mechanism in the presence of serum albumin decreases its ability to induce cytotoxicity in MDA-MB-231 cells

    Energy Technology Data Exchange (ETDEWEB)

    Pham, Anh; Bortolazzo, Anthony [Department of Biological Sciences, San Jose State University, San Jose, CA 95192-0100 (United States); White, J. Brandon, E-mail: Brandon.White@sjsu.edu [Department of Biological Sciences, San Jose State University, San Jose, CA 95192-0100 (United States)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Quercetin cannot be detected intracellularly despite killing MDA-MB-231 cells. Black-Right-Pointing-Pointer Quercetin forms a heterodimer through oxidation in media with serum. Black-Right-Pointing-Pointer The quercetin heterodimer does not kill MDA-MB-231 cells. Black-Right-Pointing-Pointer Ascorbic acid stabilizes quercetin increasing cell death in quercetin treated cells. Black-Right-Pointing-Pointer Quercetin, and not a modified form, is responsible for apoptosis and cell death. -- Abstract: Quercetin is a member of the flavonoid family and has been previously shown to have a variety of anti-cancer activities. We and others have reported anti-proliferation, cell cycle arrest, and induction of apoptosis of cancer cells after treatment with quercetin. Quercetin has also been shown to undergo oxidation. However, it is unclear if quercetin or one of its oxidized forms is responsible for cell death. Here we report that quercetin rapidly oxidized in cell culture media to form a dimer. The quercetin dimer is identical to a dimer that is naturally produced by onions. The quercetin dimer and quercetin-3-O-glucopyranoside are unable to cross the cell membrane and do not kill MDA-MB-231 cells. Finally, supplementing the media with ascorbic acid increases quercetin's ability to induce cell death probably by reduction oxidative dimerization. Our results suggest that an unmodified quercetin is the compound that elicits cell death.

  4. Cultured bovine granulosa cells rapidly lose important features of their identity and functionality but partially recover under long-term culture conditions.

    Science.gov (United States)

    Yenuganti, Vengala Rao; Vanselow, Jens

    2017-05-01

    Cell culture models are essential for the detailed study of molecular processes. We analyze the dynamics of changes in a culture model of bovine granulosa cells. The cells were cultured for up to 8 days and analyzed for steroid production and gene expression. According to the expression of the marker genes CDH1, CDH2 and VIM, the cells maintained their mesenchymal character throughout the time of culture. In contrast, the levels of functionally important transcripts and of estradiol and progesterone production were rapidly down-regulated but showed a substantial up-regulation from day 4. FOXL2, a marker for granulosa cell identity, was also rapidly down-regulated after plating but completely recovered towards the end of culture. In contrast, expression of the Sertoli cell marker SOX9 and the lesion/inflammation marker PTGS2 increased during the first 2 days after plating but gradually decreased later on. We conclude that only long-term culture conditions (>4 days) allow the cells to recover from plating stress and to re-acquire characteristic granulosa cell features.

  5. The Escherichia coli divisome: born to divide.

    Science.gov (United States)

    Natale, Paolo; Pazos, Manuel; Vicente, Miguel

    2013-12-01

    Septation in Escherichia coli involves complex molecular mechanisms that contribute to the accuracy of bacterial division. The proto-ring, a complex made up by the FtsZ, FtsA and ZipA proteins, forms at the beginning of the process and directs the assembly of the full divisome. Central to this complex is the FtsZ protein, a GTPase able to assemble into a ring-like structure that responds to several modulatory inputs including mechanisms to position the septum at midcell. The connection with the cell wall synthesising machinery stabilizes the constriction of the cytoplasmic membrane. Although a substantial amount of evidence supports this description, many details on how individual divisome elements are structured or how they function are subjected to controversial interpretations. We discuss these discrepancies arising from incomplete data and from technical difficulties imposed by the small size of bacteria. Future work, including more powerful imaging and reconstruction technologies, will help to clarify the missing details on the architecture and function of the bacterial division machinery. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  6. An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

    Directory of Open Access Journals (Sweden)

    Ying Huang

    2013-11-01

    Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

  7. A simple and rapid method for monitoring dissolved oxygen in water with a submersible microbial fuel cell (SBMFC).

    Science.gov (United States)

    Zhang, Yifeng; Angelidaki, Irini

    2012-01-01

    A submersible microbial fuel cell (SBMFC) was developed as a biosensor for in situ and real time monitoring of dissolved oxygen (DO) in environmental waters. Domestic wastewater was utilized as a sole fuel for powering the sensor. The sensor performance was firstly examined with tap water at varying DO levels. With an external resistance of 1000Ω, the current density produced by the sensor (5.6 ± 0.5-462.2 ± 0.5 mA/m(2)) increased linearly with DO level up to 8.8 ± 0.3mg/L (regression coefficient, R(2)=0.9912), while the maximum response time for each measurement was less than 4 min. The current density showed different response to DO levels when different external resistances were applied, but a linear relationship was always observed. Investigation of the sensor performance at different substrate concentrations indicates that the organic matter contained in the domestic wastewater was sufficient to power the sensing activities. The sensor ability was further explored under different environmental conditions (e.g. pH, temperature, conductivity, and alternative electron acceptor), and the results indicated that a calibration would be required before field application. Lastly, the sensor was tested with different environmental waters and the results showed no significant difference (p>0.05) with that measured by DO meter. The simple, compact SBMFC sensor showed promising potential for direct, inexpensive and rapid DO monitoring in various environmental waters. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Rapid Recovery of CD3+CD8+ T Cells on Day 90 Predicts Superior Survival after Unmanipulated Haploidentical Blood and Marrow Transplantation.

    Science.gov (United States)

    Tian, Deng-Mei; Wang, Yu; Zhang, Xiao-Hui; Liu, Kai-Yan; Huang, Xiao-Jun; Chang, Ying-Jun

    2016-01-01

    Rapid immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is significantly associated with lower infection, relapse and possibly secondary malignancy rates. The aim of this study was to investigate the role of peripheral lymphocyte subsets, especially CD3+CD8+ cytotoxic T cell recovery, in predicting transplant outcomes, including the overall survival (OS) and non-relapse mortality (NRM) rates after unmanipulated haploidentical blood and marrow transplantation (HBMT). Peripheral blood samples were obtained from 214 HBMT recipients with hematological malignancies. The peripheral lymphocyte subsets (CD3+ T cells, CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells, and CD19+ B cells) were analyzed by flow cytometry at days 30, 60, 90, 180, 270 and 360 after HBMT. The CD3+CD8+ cytotoxic T cell recovery at day 90 (CD3+CD8+-90) was correlated with bacterial infection (P = 0.001), NRM (P = 0.001), leukemia-free survival (LFS, P = 0.005), and OS (P = 0.001) at a cutoff value of 375 cells/μL CD3+CD8+ T cells. The incidence of bacterial infection in patients with the CD3+CD8+-90 at ≥375 cells/μL was significantly lower than that of cases with the CD3+CD8+-90 at CD3+CD8+ T cells at day 90 after HBMT was strongly associated with a lower incidence of NRM (HR = 0.30; 95% CI: 0.15-0.60; P = 0.000) and superior LFS (HR = 0.51; 95% CI: 0.32-0.82; P = 0.005) and OS (HR = 0.38; 95% CI: 0.23-0.63; P = 0.000). The results suggest that the rapid recovery of CD3+CD8+ cytotoxic T cells at day 90 following HBMT could predict superior transplant outcomes.

  9. Rapid Changes of mRNA-binding Protein Levels following Glucose and 3-Isobutyl-1-methylxanthine Stimulation of Insulinoma INS-1 Cells *S⃞

    Science.gov (United States)

    Süss, Christin; Czupalla, Cornelia; Winter, Christof; Pursche, Theresia; Knoch, Klaus-Peter; Schroeder, Michael; Hoflack, Bernard; Solimena, Michele

    2009-01-01

    Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic β-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the β-cell model INS-1 cells with 25 mm glucose + 1 mm IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in β-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5′- and 3′-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated β-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents. PMID:18854578

  10. A Wide-Band CMOS Injection Locked Frequency Divider

    NARCIS (Netherlands)

    Acar, M.; Leenaerts, Domine; Nauta, Bram

    In this paper we propose a novel inductorless injection-locked frequency divider (ILFD) that can make divisions with ratios 2,4,6 and 8 with wide locking ranges. Fabricated in a digital 0.18 μm CMOS process the divider can operate up to 15 GHz. The measured locking ranges of the divider for division

  11. Bridging the digital divide: with special reference to Nigeria ...

    African Journals Online (AJOL)

    This paper describes the digital divide in Africa with special reference to Nigeria. It presents the causes of digital divided such as; the general poor economic conditions in Africa; local content; telecommunications constraints; lack of investment in ICT infrastructure, etc. Benefits derivable from bridging the digital divide such ...

  12. The digital divide as a complex and dynamic phenomenon

    NARCIS (Netherlands)

    van Dijk, Johannes A.G.M.; Hacker, Kenneth

    2003-01-01

    The purpose of this article is to propose a fruitful analytical framework for data supposedly related to the concept of the socalled “digital divide.” The extent and the nature of this divide depend on the kind of access defined. Considering the possession of hardware, growing divides among

  13. Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2α.

    Science.gov (United States)

    Lee, Su Jung; Ramesh, Rashmi; de Boor, Valerie; Gebler, Jan M; Silva, Richard C; Sattlegger, Evelyn

    2017-09-01

    The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Rapid thermal processing for production of chalcopyrite thin films for solar cells: Design, analysis, and experimental implementation

    Science.gov (United States)

    Lovelett, Robert J.

    The direct conversion of solar energy to electricity, or photovoltaic energy conversion, has a number of environmental, social, and economic advantages over conventional electricity generation from fossil fuels. Currently, the most commonly-used material for photovoltaics is crystalline silicon, which is now produced at large scale and silicon-based devices have achieved power conversion efficiencies over 25% However, alternative materials, such as inorganic thin films, offer a number of advantages including the potential for lower manufacturing costs, higher theoretical efficiencies, and better performance in the field. One of these materials is the chalcopyrite Cu(InGa)(SeS) 2, which has demonstrated module efficiencies over 17% and cell efficiencies over 22%. Cu(InGa)(SeS)2 is now in the early stages of commercialization using a precursor reaction process referred to as a "selenization/sulfization" reaction. The precursor reaction process is promising because it has demonstrated high efficiency along with the large area (approximately 1 m2) uniformity that is required for modules. However, some challenges remain that limit the growth of the chalcopyrite solar cell industry including: slow reactions that limit process throughput, a limited understanding of complex reaction kinetics and transport phenomena that affect the through-film composition, and the use of highly toxic H2Se in the reaction process. In this work, I approach each of these challenges. First, to improve process throughput, I designed and implemented a rapid thermal processing (RTP) reactor, whereby the samples are heated by a 1000 W quartz-halogen lamp that is capable of fast temperature ramps and high temperature dwells. With the reactor in place, however, achieving effective temperature control in the thin film material system is complicated by two intrinsic process characteristics: (i) the temperature of the Cu(InGa)(SeS)2 film cannot be measured directly, which leaves the system without

  15. Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation

    NARCIS (Netherlands)

    Dercksen, M. W.; Gerritsen, W. R.; Rodenhuis, S.; Dirkson, M. K.; Slaper-Cortenbach, I. C.; Schaasberg, W. P.; Pinedo, H. M.; von dem Borne, A. E.; van der Schoot, C. E.

    1995-01-01

    Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very

  16. Rapid Recovery of CD3+CD8+ T Cells on Day 90 Predicts Superior Survival after Unmanipulated Haploidentical Blood and Marrow Transplantation.

    Directory of Open Access Journals (Sweden)

    Deng-Mei Tian

    Full Text Available Rapid immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT is significantly associated with lower infection, relapse and possibly secondary malignancy rates. The aim of this study was to investigate the role of peripheral lymphocyte subsets, especially CD3+CD8+ cytotoxic T cell recovery, in predicting transplant outcomes, including the overall survival (OS and non-relapse mortality (NRM rates after unmanipulated haploidentical blood and marrow transplantation (HBMT.Peripheral blood samples were obtained from 214 HBMT recipients with hematological malignancies. The peripheral lymphocyte subsets (CD3+ T cells, CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells, and CD19+ B cells were analyzed by flow cytometry at days 30, 60, 90, 180, 270 and 360 after HBMT.The CD3+CD8+ cytotoxic T cell recovery at day 90 (CD3+CD8+-90 was correlated with bacterial infection (P = 0.001, NRM (P = 0.001, leukemia-free survival (LFS, P = 0.005, and OS (P = 0.001 at a cutoff value of 375 cells/μL CD3+CD8+ T cells. The incidence of bacterial infection in patients with the CD3+CD8+-90 at ≥375 cells/μL was significantly lower than that of cases with the CD3+CD8+-90 at <375 cells/μL after HBMT (14.6% versus 41.6%, P<0.001. Multivariate analysis showed the rapid recovery of CD3+CD8+ T cells at day 90 after HBMT was strongly associated with a lower incidence of NRM (HR = 0.30; 95% CI: 0.15-0.60; P = 0.000 and superior LFS (HR = 0.51; 95% CI: 0.32-0.82; P = 0.005 and OS (HR = 0.38; 95% CI: 0.23-0.63; P = 0.000.The results suggest that the rapid recovery of CD3+CD8+ cytotoxic T cells at day 90 following HBMT could predict superior transplant outcomes.

  17. Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

    Science.gov (United States)

    Bockstael, Olivier; Melas, Catherine; Pythoud, Catherine; Levivier, Marc; McCarty, Douglas; Samulski, R Jude; De Witte, Olivier; Tenenbaum, Liliane

    2012-07-01

    The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.

  18. A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins.

    Science.gov (United States)

    Banerjee, Pratik; Lenz, Dominik; Robinson, Joseph Paul; Rickus, Jenna L; Bhunia, Arun K

    2008-02-01

    Cell-based biosensors (CBBs) are becoming important tools for biosecurity applications and rapid diagnostics in food microbiology for their unique capability of detecting physiologically hazardous materials. A multi-well plate-based biosensor containing B-cell hybridoma, Ped-2E9, encapsulated in type I collagen matrix, was developed for rapid detection of viable cells of pathogenic Listeria, the toxin listeriolysin O, and the enterotoxin from Bacillus species. This sensor measures the alkaline phosphatase release from infected Ped-2E9 cells colorimetrically. Pathogenic L. monocytogenes cells and toxin preparations from L. monocytogenes or B. cereus showed cytotoxicity ranging from 24 to 98% at 3-6 h postinfection. In contrast, nonpathogenic L. innocua (F4247) and B. subtilis induced minimal cytotoxicity, ranging only 0.4-7.6%. Laser scanning cytometry and cryo-nano scanning electron microscopy confirmed the live or dead status of the infected Ped-2E9 cells in gel matrix. This paper presents the first example of a cell-based sensing system using collagen-encapsulated mammalian cells for rapid detection of pathogenic bacteria or toxin, and demonstrates a potential for onsite use as a portable detection system.

  19. Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells

    OpenAIRE

    Li, Shengqiao; Yan, Yehong; Lin, Yuan; Bullens, Dominique; Rutgeerts, Omer; Goebels, Jozef; Segers, Constant; Boon, Louis; Kasran, Ahmad; Vos, Rita; Dewolf-Peeters, Christiane; Waer, Mark; Billiau, An

    2007-01-01

    Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cellindependent xenoantibody production are only partially understood, and their identi-ication will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic rec...

  20. The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.

    Science.gov (United States)

    Schulze, Katja; Lang, Imke; Enke, Heike; Grohme, Diana; Frohme, Marcus

    2015-04-17

    Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced

  1. Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.

    Science.gov (United States)

    Woods, G L; Mills, R D

    1988-06-01

    During a 4-month period, two methods for rapid detection of herpes simplex virus (HSV) were examined: (i) pretreatment of A549 cells with dexamethasone for conventional tissue culture (277 specimens) and (ii) 24-well plate centrifugation using A549 cells with and without dexamethasone pretreatment and staining with serotype-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h (153 specimens). By conventional tube cell culture, both with and without dexamethasone, HSV was identified in 88 of 277 (32%) specimens. Significantly more specimens were positive for HSV at 24 h (46 versus 27 specimens) and at 48 h (a total of 72 versus 59 specimens) (P less than 0.0001) in dexamethasone-treated A549 cells. Of the 153 specimens tested by conventional culture and 24-well plate centrifugation, HSV was detected in 44 (29%) by conventional culture, and by 24-well plate centrifugation with and without dexamethasone, HSV was detected in 32 (21%) and 30 (20%) specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of 24-well plate centrifugation with A549 cells for detection of HSV were 73 (71% without dexamethasone), 100, 100, and 90%, respectively. In conventional tube cell culture, pretreatment of A549 cells with dexamethasone results in more rapid detection of HSV. Centrifugal inoculation of dexamethasone-treated and untreated A549 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means to detect HSV in clinical specimens and should not replace conventional tube cell culture.

  2. A rapid and sensitive GC-MS/MS method to measure deuterium labeled deoxyadenosine in DNA from limited mouse cell populations

    Science.gov (United States)

    Farthing, Don E.; Buxbaum, Nataliya P.; Bare, Catherine V.; Treadwell, Shirin M.; Kapoor, Veena; Williams, Kirsten M.; Gress, Ronald E.

    2013-01-01

    A rapid and sensitive GC-MS/MS method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g. dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multi-parameter fluorescence activated cell sorting. Cell lysis, DNA extraction and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid on-line hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25–20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25–20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20,000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and non-relapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g. T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, HIV/AIDS, arthritis, inflammatory bowel disease, and multiple sclerosis. PMID:23541182

  3. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

    Science.gov (United States)

    Cousins, Fiona L; Murray, Alison; Esnal, Arantza; Gibson, Douglas A; Critchley, Hilary O D; Saunders, Philippa T K

    2014-01-01

    In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These

  4. Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning

    KAUST Repository

    Feizi, Alborz

    2016-09-24

    Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm2. This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP\\'s performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 105 to 1.4 × 106 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

  5. Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning.

    Science.gov (United States)

    Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

    2016-11-01

    Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm2. This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 105 to 1.4 × 106 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

  6. Cytotoxic T-lymphocyte antigen 4 blockade augments the T-cell response primed by attenuated Listeria monocytogenes resulting in more rapid clearance of virulent bacterial challenge.

    Science.gov (United States)

    Rowe, Jared H; Johanns, Tanner M; Ertelt, James M; Lai, Joseph C; Way, Sing Sing

    2009-09-01

    Cytotoxic T-lymphocyte antigen 4 (CTLA-4) uniformly suppresses antigen-specific T cells during chronic infection with bacterial, parasitic or viral pathogens. However, the importance of CTLA-4 in controlling the T-cell response during acute infection or after priming with live attenuated vaccine vectors has not been well characterized. Since strategies aimed at blocking CTLA-4 are being actively developed to therapeutically augment T-cell-mediated immunity, the effects of CTLA-4 blockade on T-cell activation during these conditions need to be more clearly defined. We have examined the role of CTLA-4 in a prime-challenge model of acute bacterial infection using both attenuated and virulent strains of the intracellular bacterium Listeria monocytogenes. Although Foxp3(+) CD4(+) T cells are the predominant CTLA-4-expressing cell type in naïve mice, antigen-specific Foxp3(-) CD4(+) cells upregulate CTLA-4 expression after primary L. monocytogenes infection. Blockade of CTLA-4 results in increased numbers of L. monocytogenes-specific CD4 and CD8 T cells after primary infection with attenuated L. monocytogenes, and confers more rapid bacterial clearance after secondary challenge with virulent L. monocytogenes. Accordingly, CTLA-4 plays an important suppressive role in T-cell priming and protective immunity in a prime-challenge model of acute bacterial infection.

  7. Airway-Resident Memory CD8 T Cells Provide Antigen-Specific Protection against Respiratory Virus Challenge through Rapid IFN-γ Production.

    Science.gov (United States)

    McMaster, Sean R; Wilson, Jarad J; Wang, Hong; Kohlmeier, Jacob E

    2015-07-01

    CD8 airway resident memory T (TRM) cells are a distinctive TRM population with a high turnover rate and a unique phenotype influenced by their localization within the airways. Their role in mediating protective immunity to respiratory pathogens, although suggested by many studies, has not been directly proven. This study provides definitive evidence that airway CD8 TRM cells are sufficient to mediate protection against respiratory virus challenge. Despite being poorly cytolytic in vivo and failing to expand after encountering Ag, airway CD8 TRM cells rapidly express effector cytokines, with IFN-γ being produced most robustly. Notably, established airway CD8 TRM cells possess the ability to produce IFN-γ faster than systemic effector memory CD8 T cells. Furthermore, naive mice receiving intratracheal transfer of airway CD8 TRM cells lacking the ability to produce IFN-γ were less effective at controlling pathogen load upon heterologous challenge. This direct evidence of airway CD8 TRM cell-mediated protection demonstrates the importance of these cells as a first line of defense for optimal immunity against respiratory pathogens and suggests they should be considered in the development of future cell-mediated vaccines. Copyright © 2015 by The American Association of Immunologists, Inc.

  8. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    Science.gov (United States)

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  9. Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.

    Science.gov (United States)

    Evans, M J; Hartman, S L; Wolff, D W; Rollins, S A; Squinto, S P

    1995-07-17

    Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed. To examine the properties of chimeric Fab derived from these monoclonal antibodies, we have developed an expression system which allows the rapid production of milligram quantities of chimeric Fab. Both the chimeric light chain and the chimeric Fd were co-expressed from the same vector, pAPEX-3P. This vector contains the SV40 origin of replication, which allows the rapid production of chimeric Fab in COS cells for preliminary characterization. Additionally, pAPEX-3P contains the Epstein-Barr virus origin of replication and a puromycin selectable marker for maintenance as a stable episome in human cell lines. A production system consisting of transfected 293-EBNA cells cultured in serum free medium followed by protein G-Sepharose chromatography of the conditioned medium was found to be sufficient for the rapid production of purified chimeric Fab. Here we have utilized this expression system to demonstrate that an anti-human C5 chimeric Fab was a potent inhibitor of complement activation in both in vitro activation assays and an ex vivo model of complement-mediated tissue damage.

  10. Sex and estrous cycle-dependent rapid protein kinase signaling actions of estrogen in distal colonic cells.

    LENUS (Irish Health Repository)

    O'Mahony, Fiona

    2008-10-01

    Previous studies from our laboratory demonstrated that 17beta-estradiol (E2) rapidly inhibits Cl(-) secretion in rat and human distal colonic epithelium. The inhibition has been shown to occur via targeting of a basolateral K(+) channel identified as the KCNQ1 (KvLQT1) channel. E2 indirectly modulates the channel activity via a cascade of second messengers which are rapidly phosphorylated in response to E2. The anti-secretory mechanism may be the manner by which E2 induces fluid retention in the intestine during periods of high circulating plasma E2. Here we review the sex-dependent and estrous cycle regulation of this novel rapid response to E2. The inhibition of KCNQ1 channel activity and Cl(-) secretion will be of interest in the future in the investigation of the retentive effects of estrogen in female tissue and also in the study of secretory disorders and drugable targets of the intestine.

  11. Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning

    Science.gov (United States)

    Fang, Yu; Frampton, John P.; Raghavan, Shreya; Sabahi-Kaviani, Rahman; Luker, Gary

    2012-01-01

    The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells. PMID:22356298

  12. Preexisting high frequencies of memory CD8+ T cells favors rapid memory differentiation and preservation of proliferative potential upon boosting

    OpenAIRE

    Fraser, Kathryn A.; Schenkel, Jason M.; Jameson, Stephen C.; Vezys, Vaiva; Masopust, David

    2013-01-01

    Memory CD8+ T cell quantity and quality determine protective efficacy against reinfection. Heterologous prime boost vaccination minimizes contraction of anamnestic effectors and maximizes memory CD8+ T cell quantity, but reportedly erodes proliferative potential and protective efficacy. This study exploited heterologous prime boost vaccination to discover parameters regulating effector CD8+ T cell contraction and memory differentiation. When abundant memory T cells were established, boosting ...

  13. Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-term engraftment in a mouse model

    NARCIS (Netherlands)

    Yeoh, J. S. G.; Ausema, A.; Wierenga, P.; de Haan, G.; van Os, R.

    In this study, we use competitive repopulation to compare the quality and frequency of stem cells isolated from mobilized blood with stem cells isolated from bone marrow (BM) in a mouse model. Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were harvested from control BM and peripheral blood of mice following

  14. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    Science.gov (United States)

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-03-04

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

  15. Rabbit antithymocyte globulin induces rapid expansion of effector memory CD8 T cells without accelerating acute graft versus host disease ?

    OpenAIRE

    Wittenbecher, Friedrich; Rieger, Kathrin; Dziubianau, Mikalai; Herholz, Anne; Mensen, Angela; Blau, Igor Wolfgang; Uharek, Lutz; Dörken, Bernd; Thiel, Andreas; Na, Il-Kang

    2013-01-01

    Rabbit antithymocyte globulin (Thymoglobulin(®)) is commonly used as graft-versus-host disease (GvHD) prophylaxis. Since we found similar total CD8 T cell numbers in patients with and without Thymoglobulin(®) therapy within the first six months after allogeneic hematopoietic stem cell transplantation, we have analyzed the reconstitution of the CD8 T cell compartment in detail. After T cell-depletion, higher and more sustained proliferative capacity of memory CD8 T cells resulted in their rapi...

  16. Rapid detection of active cytomegalovirus infection by in situ polymerase chain reaction on MRC5 cells inoculated with blood specimens.

    Science.gov (United States)

    Bettinger, D; Mougin, C; Lab, M

    1994-08-01

    An in situ polymerase chain reaction was developed to amplify immediate early genes of human cytomegalovirus in cells cultured in a 96 well plate and infected with leukocytes. The technical parameters enabling optimal detection of the DNA sequences were defined. The key to this method is the fixation of cells, which facilitates the access of the PCR mixture into the cell nuclei and preserves cell morphology. Such a technique could have wide application for the detection and identification of other infectious viruses in cultured cells very early after inoculation of clinical samples.

  17. A rapid sonication based method for preparation of stromal vascular fraction and mesenchymal stem cells from fat tissue

    Directory of Open Access Journals (Sweden)

    Mohammad Amir Amirkhani

    2016-06-01

    Conclusion: The current protocol based on the sonication-mediated cavitation is a rapid, safe and cost-effective method, which is proposed for isolation of SVF and of course ADSCs cultures in a large scale for the clinical trials or therapeutic purposes.

  18. Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC - the Hot Start experience

    Directory of Open Access Journals (Sweden)

    Paul A. De Sousa

    2017-04-01

    eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

  19. A role for prostaglandins in rapid cycling suggested by episode-specific gene expression shifts in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Gurvich, Artem; Begemann, Martin; Dahm, Liane

    2014-01-01

    OBJECTIVES: Over 12% of patients with bipolar disorder exhibit rapid cycling. The underlying biological mechanisms of this extreme form of bipolar disease are still unknown. This study aimed at replicating and extending findings of our previously published case report, where an involvement of pro...

  20. Intranasal administration of CpG induces a rapid and transient cytokine response followed by dendritic and natural killer cell activation and recruitment in the mouse lung.

    Science.gov (United States)

    Pesce, Isabella; Monaci, Elisabetta; Muzzi, Alessandro; Tritto, Elaine; Tavarini, Simona; Nuti, Sandra; De Gregorio, Ennio; Wack, Andreas

    2010-01-01

    CpG-containing oligodeoxynucleotides are potent mucosal adjuvants and effective as stand-alone treatment of respiratory infections in mice. Although CpG is also used as a type 1 helper immunomodulator in the treatment of asthma and allergic disease, immune modulation following intranasal application has not been fully characterized yet. Using a B-type CpG, we monitored RNA expression profiles, cytokine production and cellular activation in lung tissue and bronchoalveolar lavages ex vivo and cytokine production of purified cell populations in vitro. CpG triggered the upregulation of many transcripts, including interferon response genes and proinflammatory cytokine genes, between 3 h and 4 days. Overlapping subsets of these cytokine proteins were induced in vitro in purified CD11c+ cells, B cells and alveolar macrophages from the lung, thus identifying these cells as direct targets of CpG. While lung B cells strongly respond to CpG in vitro, less activation is found ex vivo, suggesting efficient CpG sequestering or rapid B cell migration after activation. In contrast, a type II alveolar epithelial cell line did not respond to CpG in vitro. We noted selective recruitment of plasmacytoid dendritic cells (DCs) into the lung tissue, and of conventional DCs and natural killer (NK) cells into the lung tissue and bronchoalveolar space. Furthermore, CpG induced activation of intrapulmonary DCs, NK and T cells. We hypothesize that CpG-linked adjuvanticity and clearance of respiratory pathogens are mediated by two major mechanisms: transient induction of the interferon pathway limiting microbial survival and selective recruitment of DCs and NK cells, which allows for better adaptive responses.

  1. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

    Science.gov (United States)

    Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

    2016-11-18

    The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

  2. Measurement of Rapid Amiloride-Dependent pH Changes at the Cell Surface Using a Proton-Sensitive Field-Effect Transistor

    Science.gov (United States)

    Schaffhauser, Daniel; Fine, Michael; Tabata, Miyuki; Goda, Tatsuro; Miyahara, Yuji

    2016-01-01

    We present a novel method for the rapid measurement of pH fluxes at close proximity to the surface of the plasma membrane in mammalian cells using an ion-sensitive field-effect transistor (ISFET). In conjuction with an efficient continuous superfusion system, the ISFET sensor was capable of recording rapid changes in pH at the cells’ surface induced by intervals of ammonia loading and unloading, even when using highly buffered solutions. Furthermore, the system was able to isolate physiologically relevant signals by not only detecting the transients caused by ammonia loading and unloading, but display steady-state signals as would be expected by a proton transport-mediated influence on the extracellular proton-gradient. Proof of concept was demonstrated through the use of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a small molecule inhibitor of sodium/hydrogen exchangers (NHE). As the primary transporter responsible for proton balance during cellular regulation of pH, non-electrogenic NHE transport is notoriously difficult to detect with traditional methods. Using the NHE positive cell lines, Chinese hamster ovary (CHO) cells and NHE3-reconstituted mouse skin fibroblasts (MSF), the sensor exhibited a significant response to EIPA inhibition, whereas NHE-deficient MSF cells were unaffected by application of the inhibitor. PMID:27043644

  3. 37 CFR 2.87 - Dividing an application.

    Science.gov (United States)

    2010-07-01

    .... (1) When the International Bureau of the World Intellectual Property Organization notifies the Office... COMMERCE RULES OF PRACTICE IN TRADEMARK CASES Classification § 2.87 Dividing an application. (a... accordance with paragraph (d) of this section. (b) Fee. In the case of a request to divide out one or more...

  4. The Digital Divide and University Education Systems in Sub ...

    African Journals Online (AJOL)

    This paper examined the concept of the digital divide, how the divide can relegate Africa to the background and how a transformed tertiary educational system can help to minimise the bridge between the information-rich countries of the North and the information-poor African countries. It is argued that if the barriers to ...

  5. Digital Natives and Digital Divide: Analysing Perspective for Emerging Pedagogy

    Science.gov (United States)

    Onye, Uriel U.; Du, Yunfei

    2016-01-01

    This paper addresses the concepts of digital natives and digital divide from the perspective of the digital outsiders (part of digital natives). It takes a critical look at the implications of available ICT in both developed and underdeveloped countries in the fight against digital divide. The major contribution to literature is by drawing…

  6. 7 CFR 800.163 - Divided-lot certificates.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Divided-lot certificates. 800.163 Section 800.163... Certificates § 800.163 Divided-lot certificates. (a) General. When shiplot grain is offered for inspection or Class X weighing as a single lot and is certificated as a single lot, the applicant may exchange the...

  7. 7 CFR 868.74 - Divided-lot certificates.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Divided-lot certificates. 868.74 Section 868.74... FOR CERTAIN AGRICULTURAL COMMODITIES Regulations Official Certificates § 868.74 Divided-lot certificates. (a) General. When commodities are offered for inspection and are certificated as a single lot...

  8. Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture.

    Science.gov (United States)

    Qin, Qi Wei; Gin, Karina Yew-Hoong; Lee, Li Yen; Gedaria, Alice Ilaya; Zhang, Sheng

    2005-04-01

    A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.

  9. The effects of the smart environment on the information divide experienced by people with disabilities.

    Science.gov (United States)

    Nam, Su-Jung; Park, Eun-Young

    2017-04-01

    Information and Communication Technology (ICT) is connected with every aspect of social, cultural, economic, educational, and commercial activity. Smart devices in particular have changed society and are necessary goods for modern people. Smart device usage is rapidly growing in everyday life, so the ability to use a smart device is increasingly important, yet there is little data supporting increased digital inclusion of people with disabilities in mobile device use. This study investigates the effects of the smart environment on the information divide experienced by people with disabilities. Data from the 2013 Information Divide Index Data of the National Information Society Agency was analyzed regarding three aspects: access, skill, and competence. The accessibility difference was investigated by comparing access to a PC or smart device in two groups. The effects of a smart environment on the information divide were analyzed using General Linear Modeling (GLM). The access rate was higher for the general group than for that of those with disabilities, and this difference appeared to be greater in the smart environment. The results of the GLM showed that disability and device access had statistically significant effects on skill and all aspects of competence. These results provide evidence that the smart environment further creates the information divide for people with disabilities. Strategies should be formed to reduce this divide, particularly within smart environments. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Metagenomics and single-cell genomics reveal high abundance of comammox Nitrospira in a rapid gravity sand filter treating groundwater

    DEFF Research Database (Denmark)

    Palomo, Alejandro; Fowler, Jane; Gülay, Arda

    genus was recovered harboring metabolic capacity for complete ammonia oxidation. We developed a cell extraction strategy that enables the disruption of Nitrospira cell clusters attached to the mineral coating of the sand. Individual cells were identified via fluorescent in situ hybridization (FISH......) with Nitrospira-specific 16S rRNA probes and sorted via fluorescence-activated cell sorting (FACS). Sorted cells were screened and selected Nitrospira spp. were subject to whole-genome sequencing. The single cell genomes confirmed the genomic presence of a complete ammonia oxidation pathway and revealed clear...... taxonomic differences with the recently described comammox Nitrospira genomes. The high abundance of comammox Nitrospira spp. together with the low abundance of canonical ammonia oxidizing prokaryotes in the investigated RSF system suggests the essential role of this novel comammox Nitrospira in the RSFs...

  11. Rapid proliferation and differentiation impairs the development of memory CD8+ T cells in early life1

    OpenAIRE

    Smith, Norah L.; Wissink, Erin; Wang, Jocelyn; Pinello, Jennifer F.; Davenport, Miles P.; Grimson, Andrew; Rudd, Brian D.

    2014-01-01

    Neonates often generate incomplete immunity against intracellular pathogens, although the mechanism of this defect is poorly understood. An important question is whether the impaired development of memory CD8+ T cells in neonates is due to an immature priming environment or lymphocyte-intrinsic defects. Here we show that neonatal and adult CD8+ T cells adopted different fates when responding to equal amounts of stimulation in the same host. While adult CD8+ T cells differentiated into a heter...

  12. [Design of broadband power divider in microwave hyperthermia system].

    Science.gov (United States)

    Sun, Bing; Jiang, Guotai; Lu, Xiaofeng; Cao, Yi

    2010-10-01

    In clinical application of microwave hyperthermia, multi-applicators are often simultaneously required to irradiate the tumor because of its large volume or its deep location. Power divider separates the input microwave energy into equal, or unequal, energy to each applicator. In this paper, the design procedure for the three-section transmission-line transformer based one-to-two equal-split Wilkinson power divider is introduced. By impedance analysis on equivalent scheme, the design parameter of power divider is provided, and by simulation and optimization on Ansoft HFSS, a microstrip structure Wilkinson power divider operating frequency 2. 45 GHz is given. Measurement test results from network analyzer show that it has 25% bandwidth and good isolation in output with this structure. Besides, it is characterized by small size and easy processing. This power divider suits microwave hyperthermia.

  13. CD4 depletion in SIV-infected macaques results in macrophage and microglia infection with rapid turnover of infected cells.

    Directory of Open Access Journals (Sweden)

    Luca Micci

    2014-10-01

    Full Text Available In rhesus macaques (RMs, experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i set-point viral load two-logs higher than controls; (ii macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii substantial expansion of pro-inflammatory monocytes; (iv aberrant activation and infection of microglial cells; and (v lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

  14. ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

    Directory of Open Access Journals (Sweden)

    Jarom Heijmans

    2013-04-01

    Full Text Available Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER stress and activity of the unfolded protein response (UPR are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

  15. Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

    Science.gov (United States)

    Schmidt, Julia; Iversen, Astrid K. N.; Tenzer, Stefan; Gostick, Emma; Price, David A.; Lohmann, Volker; Distler, Ute; Bowness, Paul; Schild, Hansjörg; Blum, Hubert E.; Klenerman, Paul

    2012-01-01

    HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope. PMID:23209413

  16. Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells

    DEFF Research Database (Denmark)

    Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla

    2009-01-01

    and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay...

  17. Reduced mRNA expression of PTGDS in peripheral blood mononuclear cells of rapid-cycling bipolar disorder patients compared with healthy control subjects

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Peijs, Lone; Kessing, Lars Vedel

    2015-01-01

    that mRNA expression of PTGDS and AKR1C3 is deregulated in rapid-cycling disorder patients in a euthymic or current affective state compared with healthy control subjects, and that expression alters with affective states. METHODS: PTGDS and AKR1C3 mRNA expression in peripheral blood mononuclear cells......BACKGROUND: Disturbances related to the arachidonic acid cascade and prostaglandin metabolism may be involved in the pathophysiology of bipolar disorder, as supported by a recent genome-wide association study meta-analysis; however, evidence from clinical studies on a transcriptional level...... was measured in 37 rapid-cycling bipolar disorder patients and 40 age- and gender-matched healthy control subjects using reverse transcription quantitative real-time polymerase chain reaction. Repeated measurements of PTGDS and AKR1C3 mRNA expression were obtained in various affective states during 6-12 months...

  18. Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience

    NARCIS (Netherlands)

    De Sousa, Paul A.; Steeg, Rachel; Wachter, Elisabeth; Bruce, Kevin; King, Jason; Hoeve, Marieke; Khadun, Shalinee; McConnachie, George; Holder, Julie; Kurtz, Andreas; Seltmann, Stefanie; Dewender, Johannes; Reimann, Sascha; Stacey, Glyn; O'Shea, Orla; Chapman, Charlotte; Healy, Lyn; Zimmermann, Heiko; Bolton, Bryan; Rawat, Trisha; Atkin, Isobel; Veiga, Anna; Kuebler, Bernd; Serano, Blanca Miranda; Saric, Tomo; Hescheler, Jürgen; Brüstle, Oliver; Peitz, Michael; Thiele, Cornelia; Geijsen, Niels; Holst, Bjørn; Clausen, Christian; Lako, Majlinda; Armstrong, Lyle; Gupta, Shailesh K.; Kvist, Alexander J.; Hicks, Ryan; Jonebring, Anna; Brolén, Gabriella; Ebneth, Andreas; Cabrera-Socorro, Alfredo; Foerch, Patrik; Geraerts, Martine; Stummann, Tina C.; Harmon, Shawn; George, Carol; Streeter, Ian; Clarke, Laura; Parkinson, Helen; Harrison, Peter W.; Faulconbridge, Adam; Cherubin, Luca; Burdett, Tony; Trigueros, Cesar; Patel, Minal J.; Lucas, Christa; Hardy, Barry; Predan, Rok; Dokler, Joh; Brajnik, Maja; Keminer, Oliver; Pless, Ole; Gribbon, Philip; Claussen, Carsten; Ringwald, Annette; Kreisel, Beate; Courtney, Aidan; Allsopp, Timothy E.

    2017-01-01

    A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was

  19. Propane 2-nitronate is more rapidly denitrified and is more genotoxic than 2-nitropropane in cultured rat hepatoma cells.

    Science.gov (United States)

    Dalke, C; Andrae, U

    1992-07-01

    We have investigated the importance of nitronate formation from 2-nitropropane (2-NP) for the oxidative metabolism and the genotoxicity of 2-NP in 2sFou rat hepatoma cells. Treatment of the cells with 2-NP for up to 3 h resulted in the time-dependent appearance of nitrite in the culture medium and in a weak induction of DNA repair synthesis. Both nitrite formation and repair induction were markedly enhanced in cells exposed to the anionic form of 2-NP, propane 2-nitronate. These observations suggest that propane 2-nitronate, rather than 2-NP itself, is oxidized by the liver cells to yield a DNA-damaging product. The results also indicate that the nitro/nitronate equilibration in intact liver cells is slow, suggesting that nitronate formation represents the rate-limiting step in the metabolic activation of 2-NP.

  20. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    Directory of Open Access Journals (Sweden)

    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  1. Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns.

    Science.gov (United States)

    Correia Carreira, S; Spencer, J; Schwarzacher, W; Seddon, A M

    2016-06-15

    In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli Finally, low numbers of magnetically labeled E. coli bacteria (Gram-negative bacteria. Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved within 15 min. This approach could be extended to encompass the capture and concentration of specific pathogens, for example, by functionalizing magnetic nanoparticles with antibodies or small molecule probes. Copyright © 2016 Correia Carreira et al.

  2. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    Science.gov (United States)

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities. © 2014 Society for Laboratory Automation and Screening.

  3. A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas.

    Directory of Open Access Journals (Sweden)

    Haolin Liu

    Full Text Available B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.

  4. Rapid bioreduction of trivalent aurum using banana stem powder and its cytotoxicity against MCF-7 and HEK-293 cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Arunkumar, Pichaimani [Bharathidasan University, Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences (India); Vedagiri, Hemamalini [Bharathidasan University, Department of Biotechnology (India); Premkumar, Kumpati, E-mail: pkumpati@hotmail.com [Bharathidasan University, Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences (India)

    2013-03-15

    Bioreduction of metal ions for the synthesis of nanoparticles of well-defined shape and size has been a great challenge in the field of nanotechnology. In this study, we explored the reduction potential of banana stem powder (BSP) for the synthesis of gold nanoparticles (GNP). The kinetics of GNP synthesis was monitored using UV-Vis spectroscopy. The synthesized GNP was characterized using dynamic light scattering (DLS), transmission electron microscopy, and fourier transform infrared spectroscopy. In addition, the cytotoxic potential of the synthesized GNP was investigated using human breast cancer (MCF-7) and normal human embryonic kidney (HEK-293) cell lines, as evaluated by changes in cell morphology, cell viability (MTT), and metabolic activity. BSP exhibited a strong reduction of Au(III) to Au (0) at room temperature within 5 min of reaction time. The synthesized GNP was found to be spherical with an average diameter of 30 nm by DLS analysis. The cytotoxicity analysis reveals a direct dose-response relationship, indicating that the cytotoxicity increases with increasing concentrations of the GNP. Significant cytotoxicity was observed in cancer cells (MCF-7) compared to normal cells (HEK-293). Also the cellular uptake of GNP was more pronounced in MCF-7 cells than HEK-293 cells as evidenced by zeta potential, implicating the possible reason for differential cytotoxicity. Thus the present study demonstrates the importance of these unique, less time-consuming, and stable BSP-mediated GNP as potential drug delivery vehicles in the application of anticancer therapy.

  5. Germ cell transplantation using sexually competent fish: an approach for rapid propagation of endangered and valuable germlines.

    Directory of Open Access Journals (Sweden)

    Sullip K Majhi

    Full Text Available The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae, were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg and high water temperature (25 degrees C treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2-13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.

  6. Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4+ T cell activation.

    Science.gov (United States)

    Ahmed, Asma; Mukherjee, Sambuddho; Deobagkar, Mukta; Naik, Tanushree; Nandi, Dipankar

    2010-11-01

    The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H(2)O(2). In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca(2+) concentrations [Ca(2+)](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNFα and IFNγ by CD4(+) T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca(2+) ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Study of Cholesterol Repletion Effect on Nanomechanical Properties of Human Umbilical Vein Endothelial Cell Via Rapid Broadband Atomic Force Microscopy.

    Science.gov (United States)

    Yan, Bo; Ren, Juan; Liu, Yue; Huang, Huarong; Zheng, Xi; Zou, Qingze

    2017-03-01

    Abnormalities of blood cholesterol concentration are associated with increased risks for vascular disease, especially heart attacks and strokes. As one of the main lipid components of plasma membrane in all mammalian cells, cholesterol has a major impact on the mechanical properties of the membrane of endothelial cells. Although the effect of cholesterol depletion on cell mechanical properties has been studied, no results yet have been reported on quantitative investigation of cholesterol repletion effect. In this study, the cholesterol repletion effect on the nanomechanical properties of human umbilical vein endothelial cell (EA.hy926) was studied using a control-based atomic force microscope (AFM) nanomechanical measurement protocol. The viscoelasticity of EA.hy926 cells were measured over a large frequency range (0.1-100 Hz) using both constant-rate excitation force with different loading rates and a broadband excitation force. The viscoelasticity oscillation of the cell membranes under the cholesterol effect was also monitored in real-time. The experiment results showed that under the effect of cholesterol repletion, both the Young's modulus and the complex modulus of EA.hy926 cell were increased over 30%, respectively, and moreover, the amplitudes of both the elasticity oscillation and the viscosity oscillation at a period of around 200 s were increased over 70%, respectively. Therefore, this work is among the first to investigate the mechanical properties, particularly, the broadband viscoelasticity variations of EA.hy926 cells under cholesterol repletion treatment. The results revealed that cholesterol repletion may reinforce the coupling of F-actin to plasma membrane by increasing actin stability, and the cholesterol might have modified the submembrane cytoskeletal organization of EA.hy926 cell by causing the involvement of the motor protein nonmuscle myosin II.

  8. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    Directory of Open Access Journals (Sweden)

    Takemoto Daigo

    2008-06-01

    Full Text Available Abstract Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the

  9. The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

    DEFF Research Database (Denmark)

    Christiansen, Stig Hill; Zhang, Xianwei; Juul-Madsen, Kristian

    2017-01-01

    in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell...... membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic...

  10. Facile and rapid generation of large-scale microcollagen gel array for long-term single-cell 3D culture and cell proliferation heterogeneity analysis.

    Science.gov (United States)

    Guan, Zhichao; Jia, Shasha; Zhu, Zhi; Zhang, Mingxia; Yang, Chaoyong James

    2014-03-04

    Microfabricated devices are suitable for single-cell analysis due to their high throughput, compatible dimensions and controllable microenvironment. However, existing devices for single-cell culture and analysis encounter some limitations, such as nutrient depletion, random cell migration and complicated fluid shear influence. Moreover, most of the single-cell culture and analysis devices are based on 2D cell culture conditions, even though 3D cell culture methods have been demonstrated to better mimic the real cell microenvironment in vivo. To solve these problems, herein we develop a microcollagen gel array (μCGA) based approach for high-throughput long-term single-cell culture and single-cell analysis under 3D culture conditions. Type-I collagen, a well-established 3D cell culture medium, was used as the scaffold for 3D cell growth. A 2 × 2 cm PDMS chip with 10 000 μCGA units was fabricated to encapsulate thousands of single cells in less than 15 min. Single cells were able to be confined and survive in μCGA units for more than 1 month. The capability of large-scale and long-term single-cell 3D culture under open culture conditions allows us to study cellular proliferation heterogeneity and drug cytotoxicity at the single-cell level. Compared with existing devices for single-cell analysis, μCGA solves the problems of nutrient depletion and random cellular migration, avoids the influence of complicated fluid shear, and mimics the real 3D growth environment in vivo, thereby providing a feasible 3D long-term single-cell culture method for single-cell analysis and drug screening.

  11. Health literacy and the digital divide among older Americans.

    Science.gov (United States)

    Levy, Helen; Janke, Alexander T; Langa, Kenneth M

    2015-03-01

    Among the requirements for meaningful use of electronic medical records (EMRs) is that patients must be able to interact online with information from their records. However, many older Americans may be unprepared to do this, particularly those with low levels of health literacy. The purpose of the study was to quantify the relationship between health literacy and use of the Internet for obtaining health information among Americans aged 65 and older. We performed retrospective analysis of 2009 and 2010 data from the Health and Retirement Study, a longitudinal survey of a nationally representative sample of older Americans. Subjects were community-dwelling adults aged 65 years and older (824 individuals in the general population and 1,584 Internet users). Our analysis included measures of regular use of the Internet for any purpose and use of the Internet to obtain health or medical information; health literacy was measured using the Rapid Estimate of Adult Literacy in Medicine-Revised (REALM-R) and self-reported confidence filling out medical forms. Only 9.7% of elderly individuals with low health literacy used the Internet to obtain health information, compared with 31.9% of those with adequate health literacy. This gradient persisted after controlling for sociodemographic characteristics, health status, and general cognitive ability. The gradient arose both because individuals with low health literacy were less likely to use the Internet at all (OR = 0.36 [95% CI 0.24 to 0.54]) and because, among those who did use the Internet, individuals with low health literacy were less likely to use it to get health or medical information (OR = 0.60 [95% CI 0.47 to 0.77]). Low health literacy is associated with significantly less use of the Internet for health information among Americans aged 65 and older. Web-based health interventions targeting older adults must address barriers to substantive use by individuals with low health literacy, or risk exacerbating the

  12. The digital divide as a complex and dynamic phenomenon

    OpenAIRE

    van Dijk, Johannes A.G.M.; Hacker, Kenneth

    2003-01-01

    The purpose of this article is to propose a fruitful analytical framework for data supposedly related to the concept of the socalled “digital divide.” The extent and the nature of this divide depend on the kind of access defined. Considering the possession of hardware, growing divides among different categories of income, employment, education, age, and ethnicity can be proved to have existed in the 1980s and 1990s according to official American and Dutch statistics. If only by effects of sat...

  13. Impact of usability on efforts to bridge the digital divide

    CSIR Research Space (South Africa)

    Adebesin, F

    2010-11-01

    Full Text Available to Bridge the Digital Divide Funmi Adebesin Meraka Institute of the CSIR and School of Computing, UNISA P.O. Box 395 Pretoria, 0001, South Africa fadebesin@csir.co.za Paula Kotzé Meraka Institute of the CSIR and School of Computing, UNISA P.O. Box.... To address the problem, international and national initiatives are ongoing to provide access to technologies with the aim of bridging the digital divide. Many of the efforts to narrow the divide have been concerned with the provision of physical...

  14. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids

    Directory of Open Access Journals (Sweden)

    Myriam Oliveira-Rodríguez

    2016-08-01

    Full Text Available Exosomes are cell-secreted nanovesicles (40–200 nm that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

  15. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids.

    Science.gov (United States)

    Oliveira-Rodríguez, Myriam; López-Cobo, Sheila; Reyburn, Hugh T; Costa-García, Agustín; López-Martín, Soraya; Yáñez-Mó, María; Cernuda-Morollón, Eva; Paschen, Annette; Valés-Gómez, Mar; Blanco-López, Maria Carmen

    2016-01-01

    Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

  16. Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide

    National Research Council Canada - National Science Library

    Watkins, Catherine L; Schmaljohann, Dirk; Futaki, Shiroh; Jones, Arwyn T

    2009-01-01

    The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full...

  17. A rapid compatibility analysis of potential offshore sand sources for beaches of the Santa Barbara Littoral Cell

    Science.gov (United States)

    Mustain, N.; Griggs, G.; Barnard, P.L.

    2007-01-01

    The beaches of the Santa Barbara Littoral Cell, which are narrow as a result of either natural and/or anthropogenic factors, may benefit from nourishment. Sand compatibility is fundamental to beach nourishment success and grain size is the parameter often used to evaluate equivalence. Only after understanding which sand sizes naturally compose beaches in a specific cell, especially the smallest size that remains on the beach, can the potential compatibility of source areas, such as offshore borrow sites, be accurately assessed. This study examines sediments on the beach and in the nearshore (5-20m depth) for the entire Santa Barbara Littoral Cell east of Point Conception. A digital bed sediment camera, the Eyeball??, and spatial autocorrelation technique were used to determine sediment grain size. Here we report on whether nearshore sediments are comparable and compatible with beach sands of the Santa Barbara Littoral Cell. ?? 2007 ASCE.

  18. Rapid Staining of Circulating Tumor Cells in Three-Dimensional Microwell Dialysis (3D-?Dialysis) Chip

    OpenAIRE

    Cho, Wanying; Pradhan, Rangadhar; Chen, Hsin Ying; Weng, Yi-Hsuan; Chu, Hsueh Yao; Tseng, Fan-Gang; Lin, Chien-Ping; Jiang, Jeng-Kai

    2017-01-01

    The conventional techniques to detect circulating tumour cells (CTCs) are lengthy and the use of centrifugal forces in this technique may cause cell mortality. As the number of CTCs in patients is quite low, the present study aims towards a gentler diagnostic procedure so as not to lose too many CTCs during the sample preparation process. Hence, a Three-Dimensional Microwell dialysis (3D-?Dialysis) chip was designed in this study to perform gentle fluorescence-removal process by using dialysi...

  19. Rapid assessment of different oxygenic phototrophs and single-cell photosynthesis with multicolour variable chlorophyll fluorescence imaging

    DEFF Research Database (Denmark)

    Trampe, Erik Christian Løvbjerg; Kolbowski, J.; Schreiber, U.

    2011-01-01

    , red or white light. Automated sequential exposure of microscopic samples to the three excitation colours enables subsequent deconvolution of the resulting fluorescence signals and colour marking of cells with different photopigmentation, i.e., cyanobacteria, green algae, red algae and diatoms......We present a new system for microscopic multicolour variable chlorophyll fluorescence imaging of aquatic phototrophs. The system is compact and portable and enables microscopic imaging of photosynthetic performance of individual cells and chloroplasts using different combinations of blue, green...

  20. The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

    DEFF Research Database (Denmark)

    Christiansen, Stig Hill; Zhang, Xianwei; Juul-Madsen, Kristian

    2017-01-01

    in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell...... of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes....

  1. Annan warns of 'content divide' between rich and poor

    CERN Multimedia

    Dickson, D

    2003-01-01

    "The Secretary-General of the United Nations, Kofi Annan, warned today (10 December) that a "content divide" is threatening to deprive developing countries of the full benefits offered by information and communications technologies (ICTs)" (1 page)

  2. Methane Isotopes from the WAIS Divide Ice Core, Version 1

    Data.gov (United States)

    National Aeronautics and Space Administration — This data set includes methane (CH4) isotope data from the West Antarctic Ice Sheet (WAIS) Divide ice core, WDC05A, in Antarctica. The data include depth, gas age,...

  3. The information divide: publishing and access issues | Baker ...

    African Journals Online (AJOL)

    information as commodity\\". Some global and local developments in information access and the Open Access movement are highlighted, and the National Library of South Africa\\'s interim strategies to bridge the information and digital divide are identified ...

  4. Continental Divide of the United States - Direct Download

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — This map layer portrays the Continental Divide of the United States. The map layer was created by extracting Hydrologic Unit Boundary line features from an existing...

  5. Divided attention: an undesirable difficulty in memory retention.

    Science.gov (United States)

    Gaspelin, Nicholas; Ruthruff, Eric; Pashler, Harold

    2013-10-01

    How can we improve memory retention? A large body of research has suggested that difficulty encountered during learning, such as when practice sessions are distributed rather than massed, can enhance later memory performance (see R. A. Bjork & E. L. Bjork, 1992). Here, we investigated whether divided attention during retrieval practice can also constitute a desirable difficulty. Following two initial study phases and one test phase with Swahili-English word pairs (e.g., vuvi-snake), we manipulated whether items were tested again under full or divided attention. Two days later, participants were brought back for a final cued-recall test (e.g., vuvi-?). Across three experiments (combined N = 122), we found no evidence that dividing attention while practicing retrieval enhances memory retention. This finding raises the question of why many types of difficulty during practice do improve long-term retention, but dividing attention does not.

  6. Divide-and-conquer approach for the exemplar breakpoint distance

    National Research Council Canada - National Science Library

    Nguyen, C Thach; Tay, Y C; Zhang, Louxin

    2005-01-01

    .... Unfortunately, the problem is NP-hard even for the breakpoint distance. This paper proposes a divide-and-conquer approach for calculating the exemplar breakpoint distance between two genomes with multiple gene families...

  7. Rapid 'on-line' image processing as a tool in the evaluation of kinetic and morphological aspects of receptor-induced cell activation.

    Science.gov (United States)

    Theler, J M; Wollheim, C B; Schlegel, W

    1991-01-01

    Transmembrane signalling involves rapid and spatially well defined changes in cytosolic free Ca2+, [Ca2+]i. Specific technologies involving image processing permit the analysis of kinetic and morphological aspects of [Ca2+]i at the subcellular level with the fluorescent Ca2+ probe fura-2. Fluorescence excitation wavelengths (340 nm or 380 nm) are alternated in synchrony with the acquisition at video rate of images captured with an intensified CCD camera. Images are digitized, recursively filtered, divided, and displayed after calibration of the 'ratio' image into a numerical [Ca2+]i scale. The image processor IMAGINE (Synoptics Ltd., UK) permits these operations at video rate. This produces 'on-line' [Ca2+]i images in real time which are stored on video tapes for subsequent analysis. The present communication summarizes the rationale for the selection of our current technologies. A comparison with alternative solutions should highlight the particular advantages and drawbacks of our approach. The present text thus should serve as a help for investigators who try to assemble image processing tools for work in the receptor and cellular signalling field.

  8. Gamma delta T cells recognize a microbial encoded B Cell antigen to initiate a rapid antigen-specific Interleukin-17 response

    Science.gov (United States)

    Gamma delta T cells contribute uniquely to host immune defense, but the way in which they do so remains an enigma. Here we show that an algae protein, phycoerythrin (PE) is recognized by gamma delta T cells from mice, bovine and humans and binds directly to specific gamma delta T cell antigen recept...

  9. Identification of Staphylococcus aureus α-hemolysin as a protein drug that is secreted by anticancer bacteria and rapidly kills cancer cells.

    Science.gov (United States)

    Swofford, Charles A; St Jean, Adam T; Panteli, Jan T; Brentzel, Zachary J; Forbes, Neil S

    2014-06-01

    Targeted bacterial delivery of anticancer proteins has the ability to overcome therapeutic resistance in tumors that limits the efficacy of chemotherapeutics. The ability of bacteria to specifically target tumors allows for delivery of aggressive proteins that directly kill cancer cells and cannot be administered systemically. However, few proteins have been tested for this purpose. To identify effective molecules, we systematically sorted proteins that have been shown to cause mammalian cell death. The genes for five proteins were selected and cloned into Escherichia coli and Salmonella. Supernatant from cultures of the transformed bacteria was applied to flasks of MCF-7 mammary carcinoma cells to identify proteins that (1) were expressed, (2) secreted, and (3) rapidly killed cancer cells. Time-lapse images were taken to visualize mammalian cell morphology. Of the investigated proteins, α-hemolysin from Staphylococcus aureus (SAH) was the most promising because it was secreted, caused trauma to cellular membranes, and induced oncosis in 18 min. After exposure for 6 h, SAH decreased cell viability by 90%. In comparison, the positive control, Pseudomonas aeruginosa exotoxin A (PEA), required 11 days to achieve a similar effect, when administered at 3,000 times its LC50 . The maximum death rate induced by SAH was calculated to be a reduction in cell viability of 7.1% per min, which was 200-fold faster than the PEA control. Two proteins, Dermonecrotic Toxin and Phospholipase C were active when extracted from the bacterial cytoplasm but were not secreted. This investigation revealed for the first time SAH as a potent anticancer drug for delivery by bacteria because of its ability to be secreted in a fully functional form and aggressively kill cancer cells. © 2014 Wiley Periodicals, Inc.

  10. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

    Directory of Open Access Journals (Sweden)

    Wagner Shin Nishitani

    Full Text Available A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7 expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

  11. HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells.

    Science.gov (United States)

    Feig, Jonathan E; Rong, James X; Shamir, Raanan; Sanson, Marie; Vengrenyuk, Yuliya; Liu, Jianhua; Rayner, Katey; Moore, Kathryn; Garabedian, Michael; Fisher, Edward A

    2011-04-26

    HDL cholesterol (HDL-C) plasma levels are inversely related to cardiovascular disease risk. Previous studies have shown in animals and humans that HDL promotes regression of atherosclerosis. We hypothesized that this was related to an ability to promote the loss of monocyte-derived cells (CD68(+), primarily macrophages and macrophage foam cells) from plaques. To test this hypothesis, we used an established model of atherosclerosis regression in which plaque-bearing aortic arches from apolipoprotein E-deficient (apoE(-/-)) mice (low HDL-C, high non-HDL-C) were transplanted into recipient mice with differing levels of HDL-C and non-HDL-C: C57BL6 mice (normal HDL-C, low non-HDL-C), apoAI(-/-) mice (low HDL-C, low non-HDL-C), or apoE(-/-) mice transgenic for human apoAI (hAI/apoE(-/-); normal HDL-C, high non-HDL-C). Remarkably, despite persistent elevated non-HDL-C in hAI/apoE(-/-) recipients, plaque CD68(+) cell content decreased by >50% by 1 wk after transplantation, whereas there was little change in apoAI(-/-) recipient mice despite hypolipidemia. The decreased content of plaque CD68(+) cells after HDL-C normalization was associated with their emigration and induction of their chemokine receptor CCR7. Furthermore, in CD68(+) cells laser-captured from the plaques, normalization of HDL-C led to decreased expression of inflammatory factors and enrichment of markers of the M2 (tissue repair) macrophage state. Again, none of these beneficial changes were observed in the apoAI(-/-) recipients, suggesting a major requirement for reverse cholesterol transport for the beneficial effects of HDL. Overall, these results establish HDL as a regulator in vivo of the migratory and inflammatory properties of monocyte-derived cells in mouse atherosclerotic plaques, and highlight the phenotypic plasticity of these cells.

  12. Justifying the Classical-Quantum Divide of the Copenhagen Interpretation

    OpenAIRE

    Bolotin, Arkady

    2014-01-01

    Perhaps the most significant drawback, which the Copenhagen interpretation (still the most popular interpretation of quantum theory) suffers from, is the classical-quantum divide between the large classical systems that carry out measurements and the small quantum systems that they measure. So, an "ideal" alternative interpretation of quantum theory would either eliminate this divide or justify it in some reasonable way. The present paper demonstrates that it is possible to justify the classi...

  13. Food deprivation causes rapid changes in the abundance and glucidic composition of the cutaneous mucous cells of Atlantic salmon Salmo salar L.

    Science.gov (United States)

    Landeira-Dabarca, A; Álvarez, M; Molist, P

    2014-10-01

    Cutaneous mucus is the first physical and chemical barrier of fish. This slime layer is secreted by mucous cells located in the epidermis and is mainly composed of glycoproteins that have their origin in the diet. Therefore, food deprivation can potentially change the abundance and glucidic nature of skin mucous cells, thus changing the mucus properties. To test this hypothesis, we conducted an experiment with Atlantic salmon, Salmo salar L. Changes in the number and glucidic nature of epidermal mucus cells were analysed using standard techniques. The outcome of this study shows that food deprivation caused a rapid decrease in the density of epidermal mucous cells in Atlantic salmon. Lectin histochemistry revealed a change in the presence and stainability of some sugar residues in the mucous cells of unfed fish compared with fed fish. Given that the primary reason for mucus secretion in fish is for protection against infections, we speculate that the changes in the mucus properties caused by nutritional stress may affect their disease resistance. This fact is particularly important for fish that spend a period of time deprived of food, either as a part of their natural life cycle, or as part of farming practices. © 2013 John Wiley & Sons Ltd.

  14. Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.

    Science.gov (United States)

    Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

    2014-03-01

    The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  15. Co-administration of Cisplatin and Furosemide Causes Rapid and Massive Loss of Cochlear Hair Cells in Mice

    Science.gov (United States)

    Li, Yongqi; Ding, Dalian; Jiang, Haiyan; Fu, Yong

    2011-01-01

    The expanding arsenal of transgenic mice has created a powerful tool for investigating the biological mechanisms involved in ototoxicity. However, cisplatin ototoxicity is difficult to investigate in mice because of their small size and vulnerability to death by nephrotoxicity. To overcome this problem, we developed a strategy for promoting cisplatin-induced ototoxicity by coadministration of furosemide a loop diuretic. A dose–response study identified 200 mg/kg of furosemide as the optimal dose for disrupting the stria vascularis and opening the blood–ear barrier. Our analysis of stria pathology indicated that the optimal period for administering cisplatin was 1 h after furosemide treatment. Combined treatment with 0.5 mg/kg of cisplatin and 200 mg/kg furosemide resulted in only moderate loss of outer hair cells in the basal 20% of the cochlea, only mild threshold shifts and minimal loss of distortion product otoacoustic emission (DPOAE). In contrast, 1 mg/kg of cisplatin plus 200 mg/kg of furosemide resulted in a permanent 40–50 dB elevation of auditory brainstem response thresholds, almost complete elimination of DPOAE, and nearly total loss of outer hair cells. The widespread outer hair cell lesions that develop in mice treated with cisplatin plus furosemide could serve as extremely useful murine model for investigating techniques for regenerating outer hair cells, studying the mechanisms of cisplatin and furosemide ototoxicity and assessing the perceptual and electrophysiological consequences of outer hair cell loss on central auditory plasticity. PMID:21455790

  16. Effects of auxins on the production of steroidal alkaloids in rapidly proliferating tissue and cell cultures of Solanum lyratum.

    Science.gov (United States)

    Kuo, Chung-Io; Chao, Chi-Hsein; Lu, Mei-Kuang

    2012-01-01

    Solanum lyratum, a rare species, is used to treat cancer, tumours and warts. Plant cell and tissue culture of S. lyratum, producing steroidal alkaloids, could be useful supplements to natural sources. To study the production of solanine, solanidine and solasodine by adding auxin-type phytohormones including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) to cell and callus cultures of S. lyratum. Methanolic extracts were made from callus and cell cultures of S. lyratumand and analysed using RP C₁₈ HPLC with UV detection. 2,4-D-induced calli from roots led to a significant enhancement in solanine production with a value of 4.13 mg/g dry weight (DW). The maximal solanidine and solasodine levels of 6.26 and 7.69 mg/g DW were respectively obtained with IBA- and IAA-treated S. lyratum cells at concentrations of 1 and 5 mg/L. Auxins were found to be useful phytohormones for the production of steroidal alkaloids. The callus and cell culture system developed is simple and can hence be a method of production of steroidal alkaloids in S. lyratum and other Solanaceae species. Copyright © 2011 John Wiley & Sons, Ltd.

  17. Dual-patterned immunofiltration (DIF) device for the rapid efficient negative selection of heterogeneous circulating tumor cells.

    Science.gov (United States)

    Bu, Jiyoon; Kang, Yoon-Tae; Kim, Young Jun; Cho, Young-Ho; Chang, Hee Jin; Kim, Hojoong; Moon, Byung-In; Kim, Ho Gak

    2016-11-29

    The analysis of circulating tumor cells (CTCs) is an emerging field for estimating the metastatic relapse and tumor burden of cancer patients. However, the isolation of CTCs is still challenging due to their ambiguity, rarity, and heterogeneity. Here, we present an anti-CD45 antibody based dual-patterned immunofiltration (DIF) device for the enrichment of heterogeneous CTC subtypes by effective elimination of leukocytes. Our uniquely designed dual-patterned layers significantly enhance the binding chance between immuno-patterns and leukocytes due to the fluidic whirling and the increased binding sites, thus achieving superior negative selection in terms of high-throughput and high purity. From the experiments using lung cancer cells, 97.07 ± 2.79% of leukocytes were eliminated with less than 10% loss of cancerous cells at the flow rate of 1 mL h(-1). To verify the device as a potential diagnostic tool, CTCs were collected from 11 cancer patients' blood and an average of 283.3 CTC-like cells were identified while less than 1 CTC-like cells were found from healthy donors. The samples were also analyzed by immunohistochemistry and the reverse transcription polymerase chain reaction to identify their heterogeneous characteristics. These remarkable results demonstrate that the present device could help to understand the unknown properties or undiscovered roles of CTCs with a non-biased view.

  18. Hydrogel Encapsulation Facilitates Rapid-Cooling Cryopreservation of Stem Cell-Laden Core-Shell Microcapsules as Cell-Biomaterial Constructs.

    Science.gov (United States)

    Zhao, Gang; Liu, Xiaoli; Zhu, Kaixuan; He, Xiaoming

    2017-12-01

    Core-shell structured stem cell microencapsulation in hydrogel has wide applications in tissue engineering, regenerative medicine, and cell-based therapies because it offers an ideal immunoisolative microenvironment for cell delivery and 3D culture. Long-term storage of such microcapsules as cell-biomaterial constructs by cryopreservation is an enabling technology for their wide distribution and ready availability for clinical transplantation. However, most of the existing studies focus on cryopreservation of single cells or cells in microcapsules without a core-shell structure (i.e., hydrogel beads). The goal of this study is to achieve cryopreservation of stem cells encapsulated in core-shell microcapsules as cell-biomaterial constructs or biocomposites. To this end, a capillary microfluidics-based core-shell alginate hydrogel encapsulation technology is developed to produce porcine adipose-derived stem cell-laden microcapsules for vitreous cryopreservation with very low concentration (2 mol L -1 ) of cell membrane penetrating cryoprotective agents (CPAs) by suppressing ice formation. This may provide a low-CPA and cost-effective approach for vitreous cryopreservation of "ready-to-use" stem cell-biomaterial constructs, facilitating their off-the-shelf availability and widespread applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Rapid allergen-induced interleukin-17 and interferon-γ secretion by skin-resident memory CD8(+) T cells

    DEFF Research Database (Denmark)

    Schmidt, Jonas D; Ahlström, Malin G; Johansen, Jeanne D

    2017-01-01

    of the challenge response correlated with the epidermal accumulation of interleukin (IL)-17A-producing and interferon (IFN)-γ-producing TRM cells. Moreover, IL-17A and IFN-γ enhanced allergen-induced IL-1β production in keratinocytes. CONCLUSIONS: We show that sensitization with contact allergens induces a strong......, long-lasting local memory and a weaker, temporary global immunological memory response to the allergen that is mediated by IL-17A-producing and IFN-γ-producing CD8(+) TRM cells....

  20. A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Hanxin Lu

    2011-02-01

    Full Text Available Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330 and HA0(1-500 proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1 as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

  1. Large-timestep techniques for particle-in-cell simulation of systems with applied fields that vary rapidly in space

    Energy Technology Data Exchange (ETDEWEB)

    Friedman, A.; Grote, D.P.

    1996-10-01

    Under conditions which arise commonly in space-charge-dominated beam applications, the applied focusing, bending, and accelerating fields vary rapidly with axial position, while the self-fields (which are, on average, comparable in strength to the applied fields) vary smoothly. In such cases it is desirable to employ timesteps which advance the particles over distances greater than the characteristic scales over which the applied fields vary. Several related concepts are potentially applicable: sub-cycling of the particle advance relative to the field solution, a higher-order time-advance algorithm, force-averaging by integration along approximate orbits, and orbit-averaging. We report on our investigations into the utility of such techniques for systems typical of those encountered in accelerator studies for heavy-ion beam-driven inertial fusion.

  2. Involvement of Ca2+ in Vacuole Degradation Caused by a Rapid Temperature Decrease in Saintpaulia Palisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue.

    Science.gov (United States)

    Ohnishi, Miwa; Kadohama, Noriaki; Suzuki, Yoshihiro; Kajiyama, Tomoharu; Shichijo, Chizuko; Ishizaki, Kimitsune; Fukaki, Hidehiro; Iida, Hidetoshi; Kambara, Hideki; Mimura, Tetsuro

    2015-07-01

    Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop. These pharmacological observations are consistent with the involvement of mechanosensitive Ca(2+) channels in the collapse of the vacuolar membrane. The high level of expression of an MCA- (Arabidopsis mechanosensitive Ca(2+) channel) like gene, a likely candidate for a mechanosensitive Ca(2+) channel(s) in plant cells, was confirmed in the palisade tissue in Saintpaulia leaves by using a newly developed method of gene expression analysis for the specialized small tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  3. Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells.

    Science.gov (United States)

    Miyata, Y; Hoshi, M; Koyasu, S; Kadowaki, T; Kasuga, M; Yahara, I; Nishida, E; Sakai, H

    1988-09-01

    Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.

  4. The development of silk fibroin scaffolds using an indirect rapid prototyping approach: morphological analysis and cell growth monitoring by spectral-domain optical coherence tomography.

    Science.gov (United States)

    Liu, M J J; Chou, S M; Chua, C K; Tay, B C M; Ng, B K

    2013-02-01

    To date, naturally derived biomaterials are rarely used in advanced tissue engineering (TE) methods despite their superior biocompatibility. This is because these native materials, which consist mainly of proteins and polysaccharides, do not possess the ability to withstand harsh processing conditions. Unlike synthetic polymers, natural materials degrade and decompose rapidly in the presence of chemical solvents and high temperature, respectively. Thus, the fabrication of tissue scaffolds using natural biomaterials is often carried out using conventional techniques, where the efficiency in mass transport of nutrients and removal of waste products within the construct is compromised. The present study identified silk fibroin (SF) protein as a suitable material for the application of rapid prototyping (RP) or additive manufacturing (AM) technology. Using the indirect RP method, via the use of a mould, SF tissue scaffolds with both macro- and micro-morphological features can be produced and qualitatively examined by spectral-domain optical coherence tomography (SD-OCT). The advanced imaging technique showed the ability to differentiate the cells and SF material by producing high contrasting images, therefore suggesting the method as a feasible alternative to the histological analysis of cell growth within tissue scaffolds. Copyright © 2011 IPEM. Published by Elsevier Ltd. All rights reserved.

  5. Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: possibility for a rapid human cell mutation assay

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, T.R.; Liber, H.L.; Penman, B.W.; Thilly, W.G.

    1978-09-29

    A thymidine kinase heterozygote designated H2BT has been isolated from the human lymphoblast line HH4. Significant increase in the trifluorothymidine-resistant fraction was observed in the new cell line following treatment with the mutagens ICR-191 and butylmethansulfonate. Phenotypic expression was complete forty-eight hours after treatment.

  6. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration.

    Science.gov (United States)

    Li, Yiqing; Andereggen, Lukas; Yuki, Kenya; Omura, Kumiko; Yin, Yuqin; Gilbert, Hui-Ya; Erdogan, Burcu; Asdourian, Maria S; Shrock, Christine; de Lima, Silmara; Apfel, Ulf-Peter; Zhuo, Yehong; Hershfinkel, Michal; Lippard, Stephen J; Rosenberg, Paul A; Benowitz, Larry

    2017-01-10

    Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn(2+)) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn(2+) increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn(2+) accumulation in amacrine cell processes involves the Zn(2+) transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn(2+) chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn(2+) chelation extends for several days after nerve injury. These results show that retinal Zn(2+) dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn(2+) chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

  7. Combinatorial Approaches to Controlling Cell Behaviour and Tissue Formation in 3D via Rapid-Prototyping and Smart Scaffold Design

    NARCIS (Netherlands)

    Woodfield, T.B.F.; Moroni, Lorenzo; Malda, Jos

    2009-01-01

    The understanding of fundamental phenomena involved in tissue engineering and regenerative medicine is continuously growing and leads to the demand for three-dimensional (3D) models that better represent tissue architecture and direct cells into the proper lineage for specific tissue repair. Porous

  8. Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics.

    Science.gov (United States)

    Sellick, Christopher A; Hansen, Rasmus; Jarvis, Roger M; Maqsood, Arfa R; Stephens, Gill M; Dickson, Alan J; Goodacre, Royston

    2010-06-15

    Fourier transform infrared (FT-IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non-secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody-producing and non-producing cell lines, and analyzed by FT-IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC-DFA), were applied to normalized FT-IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C-O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT-IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on-line measurement of antibody production in industrial scale bioreactors. 2010 Wiley Periodicals, Inc.

  9. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells

    KAUST Repository

    Ren, Jian

    2012-03-06

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E 2) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E 2 elevated [Ca 2+] i and increased Ca 2+ oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E 2 mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E 2 activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E 2 induces the non-genomic responses Ca 2+ release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E 2 responses. © 2012 Springer Science+Business Media, LLC.

  10. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells.

    Science.gov (United States)

    Ren, Jian; Wu, Jun Hua

    2012-05-01

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E(2)) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E(2) elevated [Ca(2+)]( i ) and increased Ca(2+) oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E(2) mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E(2) activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E(2) induces the non-genomic responses Ca(2+) release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E(2) responses.

  11. Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells

    Directory of Open Access Journals (Sweden)

    Ge Lin

    2009-02-01

    Full Text Available Abstract Background D-ribose in cells and human serum participates in glycation of proteins resulting in advanced glycation end products (AGEs that affect cell metabolism and induce cell death. However, the mechanism by which D-ribose-glycated proteins induce cell death is still unclear. Results Here, we incubated D-ribose with bovine serum albumin (BSA and observed changes in the intensity of fluorescence at 410 nm and 425 nm to monitor the formation of D-ribose-glycated BSA. Comparing glycation of BSA with xylose (a control for furanose, glucose and fructose (controls for pyranose, the rate of glycation with D-ribose was the most rapid. Protein intrinsic fluorescence (335 nm, Nitroblue tetrazolium (NBT assays and Western blotting with anti-AGEs showed that glycation of BSA incubated with D-ribose occurred faster than for the other reducing sugars. Protein intrinsic fluorescence showed marked conformational changes when BSA was incubated with D-ribose. Importantly, observations with atomic force microscopy showed that D-ribose-glycated BSA appeared in globular polymers. Furthermore, a fluorescent assay with Thioflavin T (ThT showed a remarkable increase in fluorescence at 485 nm in the presence of D-ribose-glycated BSA. However, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose. This suggests that glycation with D-ribose induced BSA to aggregate into globular amyloid-like deposits. As observed by Hoechst 33258 staining, 3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT and cell counting kit-8 (CCK-8 assay, lactate dehydrogenase (LDH activity assay, flow cytometry using Annexin V and Propidium Iodide staining and reactive oxygen species (ROS measurements, the amyloid-like aggregation of glycated BSA induced apoptosis in the neurotypic cell line SH-SY5Y. Conclusion Glycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important

  12. Rapid Scientific Promotion of Scientific Productions in Stem Cells According to The Indexed Papers in The ISI (web of knowledge).

    Science.gov (United States)

    Alijani, Rahim

    2015-01-01

    In recent years emphasis has been placed on evaluation studies and the publication of scientific papers in national and international journals. In this regard the publication of scientific papers in journals in the Institute for Scientific Information (ISI) database is highly recommended. The evaluation of scientific output via articles in journals indexed in the ISI database will enable the Iranian research authorities to allocate and organize research budgets and human resources in a way that maximises efficient science production. The purpose of the present paper is to publish a general and valid view of science production in the field of stem cells. In this research, outputs in the field of stem cell research are evaluated by survey research, the method of science assessment called Scientometrics in this branch of science. A total of 1528 documents was extracted from the ISI database and analysed using descriptive statistics software in Excel. The results of this research showed that 1528 papers in the stem cell field in the Web of Knowledge database were produced by Iranian researchers. The top ten Iranian researchers in this field have produced 936 of these papers, equivalent to 61.3% of the total. Among the top ten, Soleimani M. has occupied the first place with 181 papers. Regarding international scientific participation, Iranian researchers have cooperated to publish papers with researchers from 50 countries. Nearly 32% (452 papers) of the total research output in this field has been published in the top 10 journals. These results show that a small number of researchers have published the majority of papers in the stem cell field. International participation in this field of research unacceptably low. Such participation provides the opportunity to import modern science and international experience into Iran. This not only causes scientific growth, but also improves the research and enhances opportunities for employment and professional development. Iranian

  13. Understanding and mitigating the lid in SP Al-BSF Cz-Si solar cell by use of IR-belt furnace Rapid Thermal Processing

    Science.gov (United States)

    Mvutu, Mayangi Ma

    In this study, Rapid Thermal Processing was used to understand and mitigate the light-induced degradation in boron-doped Cz-Si solar cells. Two different design-types of solar cells were used in the experiment to investigate the impact the solar cell design (structure) might have in lifetime recovery. The two design types include 5-Busbar (5BB) and 4-Street-5-Busbar (4S-5BB), with a resistivity of 2 O-cm, surface area of 239 cm2, thickness of 0.018 cm, and doping concentration of 7.22 x 10 15 cm-3. In the annealing process, the peak temperature of 795°C, belt speed of 210 ipm, and dwell time of less than 2 minutes were used. It was found that solar cell design-type does not affect the lifetime recovery, since both design-types showed similar trends in all electrical output parameters. Also, although the lifetime was fully recovered following the annealing, the efficiency was not, because of contact degradation that resulted in high series resistance and junction recombination. Thus contact firing is key to effective lifetime and efficiency recovery.

  14. Mycobacterium oryzae sp. nov., a scotochromogenic, rapidly growing species is able to infect human macrophage cell line.

    Science.gov (United States)

    Ramaprasad, E V V; Rizvi, A; Banerjee, S; Sasikala, Ch; Ramana, Ch V

    2016-11-01

    Gram-stain-positive, acid-fast-positive, rapidly growing, rod-shaped bacteria (designated as strains JC290T, JC430 and JC431) were isolated from paddy cultivated soils on the Western Ghats of India. Phylogenetic analysis placed the three strains among the rapidly growing mycobacteria, being most closely related to Mycobacterium tokaiense 47503T (98.8 % 16S rRNA gene sequence similarity), Mycobacterium murale MA112/96T (98.8 %) and a few other Mycobacterium species. The level of DNA-DNA reassociation of the three strains with M. tokaiense DSM 44635T was 23.4±4 % (26.1±3 %, reciprocal analysis) and 21.4±2 % (22.1±4 %, reciprocal analysis). The three novel strains shared >99.9 % 16S rRNA gene sequence similarity and DNA-DNA reassociation values >85 %. Furthermore, phylogenetic analysis based on concatenated sequences (3071 bp) of four housekeeping genes (16S rRNA, hsp65, rpoB and sodA) revealed that strain JC290T is clearly distinct from all other Mycobacteriumspecies. The three strains had diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannosides, unidentified phospholipids, unidentified glycolipids and an unidentified lipid as polar lipids. The predominant isoprenoid quinone for all three strains was MK-9(H2). Fatty acids were C17 : 1ω7c, C16 : 0, C18 : 1ω9c, C16 : 1ω7c/C16 : 1ω6c and C19 : 1ω7c/C19 : 1ω6c for all the three strains. On the basis of phenotypic, chemotaxonomic and phylogenetic data, it was concluded that strains JC290T, JC430 and JC431 are members of a novel species within the genus Mycobacterium and for which the name Mycobacterium oryzae sp. nov. is proposed. The type strain is JC290T (=KCTC 39560T=LMG 28809T).

  15. Is there a second level divide in students Internet skills?

    DEFF Research Database (Denmark)

    Dau, Susanne; Purushothaman, Aparna

    2015-01-01

    The concept of digital divide have moved beyond physical disparities in usage and also encompasses issues impending access like human (skills) social, cultural, and psychological barriers that affect the usage of existing available technologies apart from ownership. This paper focuses on the insu......The concept of digital divide have moved beyond physical disparities in usage and also encompasses issues impending access like human (skills) social, cultural, and psychological barriers that affect the usage of existing available technologies apart from ownership. This paper focuses...... on the insufficient level of skills which students have in making the best use of available Internet technology. Varying online skills of the students in higher education from two countrywide scenarios - Denmark and India are discussed. The paper emphasizes on the reflective and conceptual issues which the students...... face to take information from the Internet that brings a second level of divide. Through a cross-national comparison the paper hopes to contribute to the literature to learn from each other´s experiences and giving insights to researchers on digital divide. The digital divide in Internet skills...

  16. Effects of total sleep deprivation on divided attention performance.

    Directory of Open Access Journals (Sweden)

    Eric Chern-Pin Chua

    Full Text Available Dividing attention across two tasks performed simultaneously usually results in impaired performance on one or both tasks. Most studies have found no difference in the dual-task cost of dividing attention in rested and sleep-deprived states. We hypothesized that, for a divided attention task that is highly cognitively-demanding, performance would show greater impairment during exposure to sleep deprivation. A group of 30 healthy males aged 21-30 years was exposed to 40 h of continuous wakefulness in a laboratory setting. Every 2 h, subjects completed a divided attention task comprising 3 blocks in which an auditory Go/No-Go task was 1 performed alone (single task; 2 performed simultaneously with a visual Go/No-Go task (dual task; and 3 performed simultaneously with both a visual Go/No-Go task and a visually-guided motor tracking task (triple task. Performance on all tasks showed substantial deterioration during exposure to sleep deprivation. A significant interaction was observed between task load and time since wake on auditory Go/No-Go task performance, with greater impairment in response times and accuracy during extended wakefulness. Our results suggest that the ability to divide attention between multiple tasks is impaired during exposure to sleep deprivation. These findings have potential implications for occupations that require multi-tasking combined with long work hours and exposure to sleep loss.

  17. Investigation on Locking and Pulling Modes in Analog Frequency Dividers

    Directory of Open Access Journals (Sweden)

    Antonio Buonomo

    2013-01-01

    Full Text Available We compare the main analytical results available to estimate the locking range, which is the key figure-of-merit of LC frequency dividers based on the injection locking phenomenon. Starting from the classical result by Adler concerning injection-locked oscillators, we elucidate the merits and the shortcomings of the different approaches to study injection-locked frequency dividers, with particular emphasis on divider-by-2. In particular, we show the potential of a perturbation approach which enables a more complete analysis of frequency dividers, making it possible to calculate not only the amplitude and the phase of the locked oscillation, but also the region where it exists and is stable, which defines the locking region. Finally, we analyze the dynamical behaviour of the dividers in the vicinity of the boundary of the locking region, showing that there exists a border region where the occurrence of the locking or the pulling operation mode is possible, depending on the initial conditions of the system.

  18. Romania's Digital Divide and the Failures of E-Government

    Directory of Open Access Journals (Sweden)

    Sorin Dan ŞANDOR

    2006-02-01

    Full Text Available This paper tries to measure the level of the digital divide existing in Romania and also to verify its relationship with e-government. At the country level, Romania is one of the least digitally developed countries in Europe, but it has shown a sharp increase in recent years. At the social level, based on data from public opinion surveys, digital divide is analyzed at two levels: lack of access and lack of knowledge. The results are similar to those in other countries: digital divide appears along the same dimensions: rural/urban, age, education, wealth and, to a lesser extent, gender. E-government services, while presumed to be at an adequate level, are used only by a very small number of citizens, thus having no impact upon the digital divide. Confronted with a serious divide, and with great possibility of its increasing, Romania needs to promote policies designed to increase access and knowledge. Also, e-government is not possible, unless administrative culture and procedures change.

  19. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.

    Science.gov (United States)

    Bressan, Raul Bardini; Dewari, Pooran Singh; Kalantzaki, Maria; Gangoso, Ester; Matjusaitis, Mantas; Garcia-Diaz, Claudia; Blin, Carla; Grant, Vivien; Bulstrode, Harry; Gogolok, Sabine; Skarnes, William C; Pollard, Steven M

    2017-02-15

    Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis. © 2017. Published by The Company of Biologists Ltd.

  20. Rapid responses to reverse T₃ hormone in immature rat Sertoli cells: calcium uptake and exocytosis mediated by integrin.

    Directory of Open Access Journals (Sweden)

    Ana Paula Zanatta

    Full Text Available There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T₃ (rT₃ action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT₃ are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT₃ was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT₃-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions. Therefore, our findings suggest that calcium uptake stimulated by rT₃ may be mediated by integrin αvβ₃. In addition, it was demonstrated that calcium uptake stimulated by rT₃ is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT₃ also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT₃ modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

  1. Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

    Science.gov (United States)

    Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

    2017-09-07

    The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  2. Rapid Fatal Acute Peripheral T-Cell Lymphoma Associated With IgG Plasma Cell Leukemia and IgA Hypergammaglobulinemia.

    Science.gov (United States)

    Jonjić, Nives; Seili Bekafigo, Irena; Fučkar Čupić, Dora; Lučin, Ksenija; Duletić Načinović, Antica; Valković, Toni

    Simultaneous occurrence of T-cell and B-cell neoplasms is rare, and etiologic relationships between these 2 malignancies are poorly understood. We describe the case of a 66-year-old woman who was admitted to the hospital because of fever, hemoptysis, lymphadenopathy, and skin rash. Enlarged lymph nodes in axillary, pectoral, paratracheal, and periportal regions as well as slight hepatomegaly and splenomegaly were confirmed. A peripheral blood smear revealed rouleaux formation and numerous circulating plasma cells, with plasmacytoid lymphocytes. Immunofixation-electrophoresis detected a monoclonal band defined as immunoglobulin (IgG)-lambda light chains with broad-band polyclonal IgA. The patient died from abrupt splenic rupture before diagnostic work-up was finished. Postmortem examination revealed infiltration of atypical lymphoid cells exhibiting high proliferative activity admixed with typical and atypical plasma cells in several organs. Thus, plasma cell leukemia (IgG-lambda) as a rare and aggressive variant of plasma cell myeloma in the present case was associated with aggressive peripheral T-cell lymphoma and polyclonal (IgA) plasmacytosis.

  3. Contacts realization by rapid thermal annealing in multicrystalline silicon solar cells with special emphasis on metal influence

    Energy Technology Data Exchange (ETDEWEB)

    El Omari, H.; Boyeaux, J.P.; Laugier, A. [Institut National des Sciences Appliquees de Lyon, Villeurbanne (France). Lab. de Physique de la Matiere

    1994-12-31

    To improve the quality of the screen printing contacts, the authors have previously shown the capability to sinter the screen printed contacts by Rapid Thermal Annealing (RTA) instead of commercial sintering; they have also noticed that the commonly used TiO{sub 2} coating deposited by spray enhances the quality of the contact either with classical annealing or RTA. The aim of the present work is to analyze the metal influence on either front or back contacts realization by RTA on Polix p type multicrystalline silicon subsequently phosphorus diffused. The screen printed contact was replaced by the chosen metal dot obtained by evaporation. The authors have studied: Al/TiO{sub 2}; Ag/Al/TiO{sub 2}; Cu/Al/TiO{sub 2}; Pt/Al/TiO{sub 2} and Cu/Cr/TiO{sub 2}. The RTA treatments were carried out at various temperatures and annealing time in an Ar ambience. The quality of the contacts are analyzed from I(V) characteristics, and possible diffusions of metallic species are characterized by SIMS experiments.

  4. Whole-cell bacterial biosensors for rapid and effective monitoring of heavy metals and inorganic pollutants in wastewater.

    Science.gov (United States)

    Olaniran, Ademola O; Hiralal, Lettisha; Pillay, Balakrishna

    2011-10-01

    The increasing number of potentially harmful pollutants in the wastewater effluent discharge necessitates the need for the development of fast and cost effective analytical techniques for extensive monitoring programmes to assess the effectiveness of the treatment process. This study compared the use of bacterial biosensors to the conventional Daphnia magna assay, Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) tests as well as chemical analysis, for monitoring the toxicity of wastewater. The bacterial biosensors constructed in this study, using S. sonnei and E. coli, were found to be sensitive to the toxicity of the wastewater effluents. A linear increase in bioluminescence with increasing concentration of heavy metals and inorganic pollutants in water was observed, with a correlation coefficient (r(2)) as high as 0.995 and 0.997, respectively. No notable correlation between biosensor toxicity and BOD and COD test results was observed. These bacterial biosensors could provide appropriate alternatives for a rapid, sensitive and cost effective detection of wastewater quality. However, the differences in sensitivity obtained for the different systems suggest that the use of a battery of toxicity assays may be required to provide a real ecotoxicological assessment of wastewater samples.

  5. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    LENUS (Irish Health Repository)

    Boehm, Manja

    2012-04-25

    AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  6. Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time).

    Science.gov (United States)

    Ross-Rodriguez, L U; Elliott, J A W; McGann, L E

    2010-08-01

    There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me(2)SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me(2)SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants. (c) 2010 Elsevier Inc. All rights reserved.

  7. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

    Directory of Open Access Journals (Sweden)

    Horváth Gábor V

    2010-01-01

    Full Text Available Abstract Background Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

  8. Rapid, long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.

    Science.gov (United States)

    Lowery, Rebecca L; Zhang, Yu; Kelly, Emily A; Lamantia, Cassandra E; Harvey, Brandon K; Majewska, Ania K

    2009-09-01

    Chronic in vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, nontoxic, and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex.

  9. BRIDGING THE INFORMATION AND DIGITAL DIVIDE AT HIGHER EDUCATIONAL INSTITUTIONS

    Directory of Open Access Journals (Sweden)

    Alla M. Kolomiiets

    2014-05-01

    Full Text Available The article deals with the notion of information and digital divide on the grounds of the world processes of the information society establishment. This paper presents a research of the level of informatization of the main spheres of life activity of citizens of foreign countries, and the analysis of the experience of the leading countries in organization and implementation of a complex of measures, aimed at crossing the digital divide. Also, there have been outlined the peculiarities and problems of the development of higher education in Ukraine in the context of information and digital inequality. On the basis of the analysis of scientific literature and personal professional experience, the ways of the solution of the problem of teacher’s selection of the adequate and the most effective pedagogical technologies, forms and methods of study in the process of overcoming the information and digital divide at higher educational institutions have been determined in this article.

  10. Echocardiographic diagnosis of divided right atrium-cor triatriatum dextrum.

    Science.gov (United States)

    Bejiqi, Ramush; Retkoceri, Ragip; Bejiqi, Hana; Zeka, Naim; Kelmendi, Myrvete

    2010-01-01

    First reported in 1868, cor triatriatum, that is, a heart with 3 atria (triatrial heart), is a congenital anomaly in which the left atrium (cor triatriatum sinistrum) or right atrium (cor triatriatum dextrum) is divided into two parts by a fold of tissue, a membrane, or a fibromuscular band. As far as division of the morphologically left atrium (cor triatriatum) is a recognized clinical and surgical entity. Division of the right atrium (prominence of the eustachian and thebesian valves) is recognized pathologically, but is rare. A partition division the right atrium was attached to the orifice of the inferior caval vei. The dividing partition is placed between the systemic venous sinus and the distal part of the right atrium, made up of the vestibule and appendage. This report describes a case of a divided right atrium, associated with a large atrial septal defect and valvular pulmonary stenosis.

  11. Selectively Distracted: Divided Attention and Memory for Important Information.

    Science.gov (United States)

    Middlebrooks, Catherine D; Kerr, Tyson; Castel, Alan D

    2017-08-01

    Distractions and multitasking are generally detrimental to learning and memory. Nevertheless, people often study while listening to music, sitting in noisy coffee shops, or intermittently checking their e-mail. The current experiments examined how distractions and divided attention influence one's ability to selectively remember valuable information. Participants studied lists of words that ranged in value from 1 to 10 points while completing a digit-detection task, while listening to music, or without distractions. Though participants recalled fewer words following digit detection than in the other conditions, there were no significant differences between conditions in terms of selectively remembering the most valuable words. Similar results were obtained across a variety of divided-attention tasks that stressed attention and working memory to different degrees, which suggests that people may compensate for divided-attention costs by selectively attending to the most valuable items and that factors that worsen memory do not necessarily impair the ability to selectively remember important information.

  12. Dividing traffic cluster into parts by signal control

    Science.gov (United States)

    Nagatani, Takashi

    2018-02-01

    When a cluster of vehicles with various speeds moves through the series of signals, the cluster breaks down by stopping at signals and results in smaller groups of vehicles. We present the nonlinear-map model of the motion of vehicles controlled by the signals. We study the breakup of a cluster of vehicles through the series of signals. The cluster of vehicles is divided into various groups by controlling the cycle time of signals. The vehicles within each group move with the same mean velocity. The breakup of the traffic cluster depends highly on the signal control. The dependence of dividing on both cycle time and vehicular speed is clarified. Also, we investigate the effect of the irregular interval between signals on dividing.

  13. Ouroboros: A Tool for Building Generic, Hybrid, Divide& Conquer Algorithms

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J R; Foster, I

    2003-05-01

    A hybrid divide and conquer algorithm is one that switches from a divide and conquer to an iterative strategy at a specified problem size. Such algorithms can provide significant performance improvements relative to alternatives that use a single strategy. However, the identification of the optimal problem size at which to switch for a particular algorithm and platform can be challenging. We describe an automated approach to this problem that first conducts experiments to explore the performance space on a particular platform and then uses the resulting performance data to construct an optimal hybrid algorithm on that platform. We implement this technique in a tool, ''Ouroboros'', that automatically constructs a high-performance hybrid algorithm from a set of registered algorithms. We present results obtained with this tool for several classical divide and conquer algorithms, including matrix multiply and sorting, and report speedups of up to six times achieved over non-hybrid algorithms.

  14. A new general and rapid method for investigating hot corrosion: preliminary tests on electrodes for molten carbonate fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Griffiths, T.R. [Dept. of Chemistry, Univ. of Leeds, Leeds (United Kingdom); Volkovich, V.A. [Dept. of Rare Metals, Ural State Technical Univ. - UPI, Ekaterinburg (Russian Federation)

    2004-07-01

    With the increase and improvement in corrosion resistant alloys, coatings and surface treatments, the ability to test and predict their long-term behaviour to oxidation under hot corrosion conditions, within a reasonable time period, becomes diminished. Peroxide and superoxide ions are the most active oxidisers and we have established a means for generating them in significant concentrations in molten carbonates. This thus has the potential for enabling accelerated hot corrosion studies and we here describe the technique and report preliminary studies on the nickel and nickel oxide electrodes used in molten carbonate fuel cells. Further applications are expected to follow. (orig.)

  15. Newly divided eosinophils limit ozone-induced airway hyperreactivity in nonsensitized guinea pigs.

    Science.gov (United States)

    Wicher, Sarah A; Jacoby, David B; Fryer, Allison D

    2017-06-01

    Ozone causes vagally mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs, where they mediate ozone-induced hyperreactivity 1 day after exposure but are paradoxically protective 3 days later. We aimed to test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and nonsensitized guinea pigs. Nonsensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 h. Later (1 or 3 days later), vagally induced bronchoconstriction was measured, and inflammatory cells were harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day after ozone, mature eosinophils dominate the inflammatory response and potentiate vagally induced bronchoconstriction. However, by 3 days, newly divided eosinophils have reached the lungs, where they inhibit ozone-induced airway hyperreactivity because depleting them with antibody to IL-5 or a TNF-α antagonist worsened vagally induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs 3 days later failed to occur. Thus mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs, where 3 days later, newly divided eosinophils attenuate vagally mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNF-α antagonist or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus interventions targeting eosinophils, although beneficial in atopic individuals, may delay resolution of airway hyperreactivity in nonatopic individuals. Copyright

  16. Transformer-based design techniques for oscillators and frequency dividers

    CERN Document Server

    Luong, Howard Cam

    2016-01-01

    This book provides in-depth coverage of transformer-based design techniques that enable CMOS oscillators and frequency dividers to achieve state-of-the-art performance.  Design, optimization, and measured performance of oscillators and frequency dividers for different applications are discussed in detail, focusing on not only ultra-low supply voltage but also ultra-wide frequency tuning range and locking range.  This book will be an invaluable reference for anyone working or interested in CMOS radio-frequency or mm-Wave integrated circuits and systems.

  17. Enhanced Model of Nonlinear Spiral High Voltage Divider

    Directory of Open Access Journals (Sweden)

    V. Panko

    2015-04-01

    Full Text Available This paper deals with the enhanced accurate DC and RF model of nonlinear spiral polysilicon voltage divider. The high resistance polysilicon divider is a sensing part of the high voltage start-up MOSFET transistor that can operate up to 700 V. This paper presents the structure of a proposed model, implemented voltage, frequency and temperature dependency, and scalability. A special attention is paid to the ability of the created model to cover the mismatch and influence of a variation of process parameters on the device characteristics. Finally, the comparison of measured data vs. simulation is presented in order to confirm the model validity and a typical application is demonstrated.

  18. Google and the digital divide the bias of online knowledge

    CERN Document Server

    Segev, Elad

    2010-01-01

    Beneficial to scholars and students in the fields of media and communication, politics and technology, this book outlines the significant role of search engines in general and Google in particular in widening the digital divide between individuals, organisations and states. It uses innovative methods and research approaches to assess and illustrate the digital divide by comparing the popular search queries in Google and Yahoo in different countries as well as analysing the various biases in Google News and Google Earth. The different studies developed and presented in this book provide various

  19. A mouse model for inducible overexpression of Prdm14 results in rapid-onset and highly penetrant T-cell acute lymphoblastic leukemia (T-ALL

    Directory of Open Access Journals (Sweden)

    Brandi L. Carofino

    2013-11-01

    PRDM14 functions in embryonic stem cell (ESC maintenance to promote the expression of pluripotency-associated genes while suppressing differentiation genes. Expression of PRDM14 is tightly regulated and typically limited to ESCs and primordial germ cells; however, aberrant expression is associated with tumor initiation in a wide variety of human cancers, including breast cancer and leukemia. Here, we describe the generation of a Cre-recombinase-inducible mouse model for the spatial and temporal control of Prdm14 misexpression [ROSA26 floxed-stop Prdm14 (R26PR]. When R26PR is mated to either of two Cre lines, Mx1-cre or MMTV-cre, mice develop early-onset T-cell acute lymphoblastic leukemia (T-ALL with median overall survival of 41 and 64 days for R26PR;Mx1-cre and R26PR;MMTV-cre, respectively. T-ALL is characterized by the accumulation of immature single-positive CD8 cells and their widespread infiltration. Leukemia is preceded by a dramatic expansion of cells resembling hematopoietic stem cells and lymphoid-committed progenitors prior to disease onset, accompanied by a blockage in B-cell differentiation at the early pro-B stage. Rapid-onset PRDM14-induced T-ALL requires factors that are present in stem and progenitor cells: R26