Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Effects of the aurora kinase inhibitors AZD1152-HQPA and ZM447439 on growth arrest and polyploidy in acute myeloid leukemia cell lines and primary blasts.

BACKGROUND:
Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents.
DESIGN AND METHODS:
Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples.
RESULTS:
The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies.
CONCLUSIONS:
AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.

Ca(2+) signals coordinate zygotic polarization and cell cycle progression in the brown alga Fucus serratus

Zygotes of the fucoid brown algae provide excellent models for addressing fundamental questions about zygotic symmetry breaking. Although the acquisition of polarity is tightly coordinated with the timing and orientation of the first asymmetric division-with zygotes having to pass through a G1/S-phase checkpoint before the polarization axis can be fixed -the mechanisms behind the interdependence of polarization and cell cycle progression remain unclear. In this study, we combine in vivo Ca(2+) imaging, single cell monitoring of S-phase progression and multivariate analysis of high-throughput intracellular Ca(2+) buffer loading to demonstrate that Ca(2+) signals coordinate polarization and cell cycle progression in the Fucus serratus zygote. Consistent with earlier studies on this organism, and in contrast to animal models, we observe no fast Ca(2+) wave following fertilization. Rather, we show distinct slow localized Ca(2+) elevations associated with both fertilization and S-phase progression, and we show that both S-phase and zygotic polarization are dependent on pre-S-phase Ca(2+) increases. Surprisingly, this Ca(2+) requirement cannot be explained by co-dependence on a single G1/ S-phase checkpoint, as S phase and zygotic polarization are differentially sensitive to pre-S-phase Ca(2+) elevations and can be uncoupled. Furthermore, subsequent cell cycle progression through M phase is independent of localized actin polymerization and zygotic polarization. This absence of a morphogenesis checkpoint, together with the observed Ca(2+)dependences of S phase and polarization, show that the regulation of zygotic division in the brown algae differs from that in other eukaryotic model systems, such as yeast and Drosophila.

A tubular direct methanol fuel cell with Ti mesh anode

A novel tubular cell structure for a direct methanol fuel cell (DMFC) is proposed based on a tubular Ti mesh and a Ti mesh anode. A dip coating method has been developed to fabricate the cell. The characterization of the tubular MEA has been analyzed by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), half cell and single cell testing. The tubular DMFC single cell comprises: a Ti mesh, a cathode diffusion layer and catalyst layer, a Nafion recast membrane and a PtRuO/Ti anode. Half cell tests show that the optimum catalyst loading, Ru/(Ru + Pt) atomic ratio and the Nafion loading of a PtRuO/Ti mesh anode are: 4 mg cm, 38% and 0.6 mg cm, respectively. Single cell tests show that the Nafion loading of the recast Nafion membrane and the concentration of the methanol in the electrolyte have a major influence on cell performance. © 2006 Elsevier B.V. All rights reserved.

The impacts of replacing air bubbles with microspheres for the clarification of algae from low cell-density culture

Dissolved Air Flotation (DAF) is a well-known coagulation-flotation system applied at large scale for microalgae harvesting. Compared to conventional harvesting technologies DAF allows high cell recovery at lower energy demand. By replacing microbubbles with microspheres, the innovative Ballasted Dissolved Air Flotation (BDAF) technique has been reported to achieve the same algae cell removal efficiency, while saving up to 80% of the energy required for the conventional DAF unit. Using three different algae cultures (Scenedesmus obliquus, Chlorella vulgaris and Arthrospira maxima), the present work investigated the practical, economic and environmental advantages of the BDAF system compared to the DAF system. 99% cells separation was achieved with both systems, nevertheless, the BDAF technology allowed up to 95% coagulant reduction depending on the algae species and the pH conditions adopted. In terms of floc structure and strength, the inclusion of microspheres in the algae floc generated a looser aggregate, showing a more compact structure within single cell alga, than large and filamentous cells. Overall, BDAF appeared to be a more reliable and sustainable harvesting system than DAF, as it allowed equal cells recovery reducing energy inputs, coagulant demand and carbon emissions. © 2014 Elsevier Ltd.

Phosphorylated DegU Manipulates Cell Fate Differentiation in the Bacillus subtilis Biofilm

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

Time and Cell Type Dependency of Survival Responses in Co-cultured Tumor and Fibroblast Cells after Exposure to Modulated Radiation Fields

Advanced radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) achieve high levels of conformity to the target volume through the sequential delivery of highly spatially and temporally modulated radiation fields, which have been shown to impact radiobiological response. This study aimed to characterize the time and cell type dependency of survival responses to modulated fields using single cell type (SCT) and mixed cell type (MCT) co-culture models of transformed fibroblast (AG0-1522b) cells, and prostate (DU-145) and lung (H460) cancer cells. In SCT cultures, in-field responses showed no significant time dependency while out-of-field responses occurred early, and plateaued 6 h after irradiation in both DU-145 and H460 cells. Under modulated beam configurations MCT co-cultures showed cell-specific, differential out-of-field responses depending on the irradiated in-field and responding out-of-field cell type. The observed differential out-of-field responses may be due to the genetic background of the cells, in particular p53 status, which has been shown to mediate radiation-induced bystander effects (RIBEs). These data provide further insight into the radiobiological parameters that influence out-of-field responses, which have potential implications for advanced radiotherapy modalities and may provide opportunities for biophysical optimization in radiotherapy treatment planning.

Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus.

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.

Transcriptional regulation of effector CD8+ T cell differentiation and molecular dysfunction during HIV-1 infection

Les cellules T CD8+ jouent un rôle primordial dans le contrôle des infections virales en limitant la dissémination des cellules infectées. Lors de l’infection chronique par le virus HIV, les cellules T CD8+ HIV-spécifiques ne se différencient pas en cellules effectrices fonctionnelles capables de tuer les cellules infectées par le virus ; ces cellules ne sont plus capables de proliférer ou de produire l’ IL-2. Ces cellules expriment PD-1 et l’engagement de PD-1, par son ligand, aboutit a plusieurs de ces déficits fonctionnels des cellules T . Le rôle de PD-1 dans la régulation d'évènements transcriptionnels contrôlant la différentiation et l'obtention des fonction effectrices des cellules T CD8+ reste à démontrer. Id2 joue un rôle central dans la différenciation des cellules T CD8+ effectrices. Nous avons émis l’hypothèse que le défaut de maturation observé chez les cellules T CD8+ PD-1 high HIV-spécifiques (CD8+PD-1hi) au cours de l’infection chronique par le virus HIV pouvait être lié à la diminution d’expression du régulateur Id2. Nous avons ainsi démontré que l'engagement de PD-1 contribuait à une diminution d'expression de Id2 et de ses cibles transcriptionnelles. La surexpression de Id2 de ces cellules a permis de restaurer l'expression de marqueurs tels que Granzyme B et Bcl-2 et diminuir l’expression du marqueur de maturation de CD27. La famille des cytokines à chaine gamma joue un rôle clef dans la survie et l’homéostasie des cellules T. Dans ce travail, nous avons démontré que l’IL-15 était unique grâce à ses capacités de stimulation de l’expression d’Id2 et ses propriétés favorisant la survie ainsi que la différenciation des cellules T CD8+ effectrices. l’IL-15 induit la prolifération de toutes les populations de cellules T mémoires provenant de donneurs sains. L’addition de cette cytokine aux sous-populations cellulaires Ttm et Tem a permis leur différenciation en cellules effectrices capables de produire Granzyme B alors que la stimulation par l’IL-15 des cellules Tcm ne favorise pas leur différenciation. Un test de cytotoxicitié par cytométrie en flux nous a permis de confirmer que la stimulation de cellules T CD8+ HIV spécifiques par l’IL-15 favorisait l’expression de Id2 et restaurait les fonctions cytotoxiques des cellules T CD8+ HIV spécifiques. En conclusion, nous avons pour la première fois dans cette thèse défini les mécanismes moléculaires impliqués dans la modulation de l’expression du régulateur transcriptionnel Id2 par l’IL-15. Nous avons également révélé comment l’engagement de PD-1 conduisait a une altération de l’expression et de la fonction d’Id2 et favorisait la diminution des fonctions effectrices des cellules T CD8-HIV spécifiques. Une perspective de traitement avec des agents tels que l’IL-15 ou le bloquage de PD-1, en combinaison avec les traitements conventionnels, pourrait contribuer à une meilleure stimulation des réponses immunes favorisant ainsi la réactivation des cellules T CD8+ et permettant la destruction de cellules T CD4+ infectées de manière latente.

“Development of lymphoid cell culture system from Penaeus monodon and molecular approaches for its transformation

Unveiling the molecular and regulatory mechanisms that prevent in vitro transformation in shrimp remains elusive in the development of continuous cell lines, with an arduous history of over 25 years (Jayesh et al., 2012). Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. In addition, the regulatory mechanisms that prevent in vitro transformation and carcinoma in shrimps might provide new leads for the development of anti-ageing and anti-cancer interventions in human (Vogt, 2011) and in higher vertebrates. This highlights the importance of developing shrimp cell lines, to bring out effective prophylactics against shrimp viruses and for understanding the mechanism that induce cancer and ageing in human.. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. With this backdrop, the research described in this thesis attempted to develop molecular tools for induced in vitro transformation in lymphoid cells from Penaeus monodon and for the development of continuous cell lines using conventional and novel technologies to address the problems at cellular and molecular level.

Establishment of Shrimp Cell Lines: Perception and Orientation

Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed

Isolating single primary rat hippocampal neurons & astrocytes on ultra-thin patterned parylene-C/silicon dioxide substrates

We report here the patterning of primary rat neurons and astrocytes from the postnatal hippocampus on ultra-thin parylene-C deposited on a silicon dioxide substrate, following observations of neuronal, astrocytic and nuclear coverage on strips of different lengths, widths and thicknesses. Neuronal and glial growth was characterized ‘on’, ‘adjacent to’ and ‘away from’ the parylene strips. In addition, the article reports how the same material combination can be used to isolate single cells along thin tracks of parylene-C. This is demonstrated with a series of high magnification images of the experimental observations for varying parylene strip widths and thicknesses. Thus, the findings demonstrate the possibility to culture cells on ultra-thin layers of parylene-C and localize single cells on thin strips. Such work is of interest and significance to the Neuroengineering and Multi-Electrode Array (MEA) communities, as it provides an alternative insulating material in the fabrication of embedded micro-electrodes, which can be used to facilitate single cell stimulation and recording in capacitive coupling mode.

First human hNT neurons patterned on parylene-C/silicon dioxide substrates: Combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain

In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible cell line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single cell and network level.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Investigation of the CO tolerance mechanism at several Pt-based bimetallic anode electrocatalysts in a PEM fuel cell

The electrocatalysis of CO tolerance of Pt/C, PtRu/C, PtFe/C, PtMo/C, and PtW/C at a PEM fuel cell anode has been investigated using single cell polarization and online electrochemical mass spectrometry (EMS) measurements, and cyclic voltammetry, X-ray diffraction (XRD), in situ X-ray absorption near edge structure (XANES) analyses of the electrocatalysts. For all bimetallic electrocatalysts, which presented higher CO tolerance, EMS results have shown that the production of CO(2) start at lower hydrogen electrode overpotentials as compared to Pt/C, confirming the occurrence of the so-called bifunctional mechanism. On the other hand, XANES results indicate an increase in the Pt 5d-band vacancies for the bimetallic catalysts, particulary for PtFe/C, this leading to a weakening of the Pt-CO bond, helping to increase the CO tolerance (the so-called electronic effect). For PtMo/C and PtRu/C supplied with H(2)/CO, the formation of CO(2) is observed even when the cell is at open circuit, confirming some elimination of CO by a chemical process, most probably the water gas shift reaction. (C) 2008 Elsevier Ltd. All rights reserved.