Characterization of apoptosis in PER.C6® batch and perfusion cultures
Mercier, S.M. ; Diepenbroek, B. ; Martens, D.E. ; Wijffels, R.H. ; Streefland, M. - \ 2015
Biotechnology and Bioengineering 112 (2015)3. - ISSN 0006-3592 - p. 569 - 578.
hamster ovary cells - high-level expression - flow-cytometry - line per.c6 - death - adenovirus - dna - vaccine - gene - manufacture
Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimization of production processes for biopharmaceuticals. Cell cultures need to be maintained highly viable for extended times in order to reach maximum production yields. Moreover, programmed cell death through apoptosis is often believed to occur without being detected by classical viability measurements. In this study, we characterized cell death in PER.C6® batch and perfusion cultures using three flow cytometry techniques measuring different steps of the apoptosis cascade: DNA fragmentation, caspases activation and phosphatidylserine externalization. We showed that apoptosis is the main pathway of PER.C6® cell death in batch cultures after depletion of main carbon sources. In high cell density perfusion cultures fed at a constant specific perfusion rate, both high viability and very limited apoptosis were observed. When extending this perfusion process far beyond standard operations, cultures were exposed to suboptimal process conditions, which resulted in an increase of apoptotic cell death. Moreover, we showed that the reference viability measurement using trypan blue exclusion properly assesses the level of cell death in PER.C6® cultures. This study is a first step in understanding the mechanisms of PER.C6® cell death, which will be helpful to support applications of the cell line.
Interactions between formulation and spray drying conditions related to survival of Lactobacillus plantarum WCFS1
Perdana, J.A. ; Fox, M.B. ; Siwei, C. ; Boom, R.M. ; Schutyser, M.A.I. - \ 2014
Food Research International 56 (2014). - ISSN 0963-9969 - p. 9 - 17.
glass-transition temperature - membrane phase-behavior - lactic-acid bacteria - flow-cytometry - industrial applications - dairy ingredients - osmotic-stress - water activity - rhamnosus gg - gel phase
Protective solid carriers are commonly added to probiotic cultures prior to drying. Their formulation is not trivial and depends on the drying conditions applied. In this study, we systematically investigated the influence of formulation parameters on the survival of Lactobacillus plantarum WCFS1 after drying. Low molecular weight carbohydrates (less than 2 kDa) with high glass transition temperatures provided the highest level of protection at both low (25 degrees C) and high (50 degrees C or higher) drying temperatures. Low molecular weight carbohydrates may provide stabilization by closely interacting with the lipid bilayer of the cell membranes. Meanwhile, carbohydrates with high glass transition temperatures probably provide stabilization via fixation of the cells in a glassy powder. Furthermore, adequate amounts of solid carrier are required to sufficiently stabilize the cells during drying. During drying, crystallization of solid carriers may occur. Depending on the crystal geometry, crystallization can be either beneficial (e.g. with mannitol or sorbitol) or detrimental (e.g. with lactose) to cell survival. Finally, the effect of formulation on cell viability during storage was studied. A decimal reduction time of approximately 300 days was observed when spray dried L. plantarum WCFS1 was stored at temperatures below 40 degrees C. The outcome of this study was used as a basis to construct a generalized diagram to indicate the combinations of formulation and drying conditions to maximally retain viability and operate dryers at high efficiency. (C) 2013 Elsevier Ltd. All rights reserved.
F1 hybrid of cultivated apple (Malus x domestica) and European pear (Pyrus communis) with fertile F2 offspring
Fischer, T.C. ; Malnoy, M. ; Hofmann, T. ; Schwab, W. ; Palmieri, L. ; Wehrens, H.R.M.J. ; Schuch, L.A. ; Müller, M. ; Schimmelpfeng, H. ; Velasco, R. ; Martens, S. - \ 2014
Molecular Breeding 34 (2014)3. - ISSN 1380-3743 - p. 817 - 828.
nuclear-dna content - genetic-linkage maps - flow-cytometry - japanese pear - s-alleles - borkh. - diversity - rosaceae - markers - genome
The establishment of intergeneric hybrids for horticultural and agricultural crops is still a demanding task for breeding programmes. The aim of such approaches is to introduce new quality and resistance traits and to enlarge the gene pool. Recently, an F1 hybrid between Malus × domestica and Pyrus communis became available which arose from a breeding approach undertaken in the late 1980s by the breeder Max Zwintzscher (Cologne-Vogelsang). Unlike previous reports, viable and fertile F2 plants were obtained from this F1 hybrid line by author HS, providing a unique perspective not only for genomic, transcriptomic and metabolomic studies but also for advanced breeding strategies. Here, we give the first report on the confirmation and characterization of the F1 hybrid by phenotypic, genetic and biochemical means. The intergeneric hybrid shows an intermediary phenotype of leaves, flowers and fruits, and some disorder of secondary shoot growth. Nuclear DNA content is also intermediary and corresponds to a diploid state. Apple and pear type rDNA as well as SI alleles from each genus were found. At the metabolic level, parallel biosynthesis of the apple dihydrochalcone phloridzin and of arbutin, a p-hydroquinone-glucoside typical for pear, take place leading to considerable concentrations of both in leaves. The overall data allow secure confirmation of the hybrid character and give a first insight into the hybrids genetics and physiology.
Reporter-based screening and selection of enzymes
Rossum, T. van; Kengen, S.W.M. ; Oost, J. van der - \ 2013
FEBS Journal 280 (2013)13. - ISSN 1742-464X - p. 2979 - 2996.
induced gene-expression - protein-protein interactions - directed evolution - chemical complementation - computational design - metagenome libraries - in-vivo - flow-cytometry - identification - biocatalysts
The biotech industry is continuously seeking for new or improved biocatalysts. The success of these efforts is often hampered by the lack of an efficient screening assay. Thus, to be able to extend the number of enzymes available for industrial applications, high-throughput screening and selection methods are required. In the last few years an impressive range of screening and selection strategies has been developed. In this review, we will mainly focus on in vivo reporter systems in which the activity of a reporter is controlled by the activity of an enzyme of interest. Different mechanisms can be distinguished: (a) binding of the product of the enzymatic reaction to a transcriptional regulator and thereby turning on transcription of the reporter; (b) direct modification of a transcriptional regulator by the enzyme resulting in expression of the reporter; (c) binding of the product to a regulatory riboswitch or ribozyme, resulting in translation of the reporter; and (d) direct modification of the reporter by the enzyme, altering the reporter's activity. The choice for either a selection or a screening strategy depends on the type of reporter, e.g. providing antibiotic resistance (selection) or transmitting a fluorescent signal (screening). Although developing the specificity of each of these reporter-based selection or screening systems towards a certain enzymatic reaction is not yet straightforward, their adjustable modular design appears to be a promise for general applicability in the near future
Rapid Susceptibility Testing and Microcolony Analysis of Candida spp. Cultured and Imaged on Porous Aluminum Oxide
Ingham, C.J. ; Boonstra, S. ; Levels, S. ; Lange, H.J. ; Meis, J.F. ; Schneeberger, P.M. - \ 2012
PLoS ONE 7 (2012)3. - ISSN 1932-6203
antifungal drug-resistance - flow-cytometry - interpretive breakpoints - voriconazole - growth - fun-1 - microorganisms - fluconazole - albicans - support
Background: Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results. Methods: Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture). Significance: Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.
Single-cell genomics: unravelling the genomes of unculturable microorganisms
Jager, V.C.L. de; Siezen, R.J. - \ 2011
Microbial Biotechnology 4 (2011)4. - ISSN 1751-7907 - p. 431 - 437.
genetic-analysis - marine sponges - flow-cytometry - amplification - heterogeneity - communities - polymerase
Antioxidant micronutrients improve intrinsic and UV-induced apoptosis of human lymphocytes particularly in elderly people
Ma, A.G. ; Ge, S. ; Zhang, M. ; Shi, X.X. ; Schouten, E.G. ; Kok, F.J. ; Sun, Y.Y. ; Han, X.X. - \ 2011
Journal of Nutrition, Health and Aging 15 (2011)10. - ISSN 1279-7707 - p. 912 - 917.
peripheral-blood lymphocytes - oxidative stress - cell-death - flow-cytometry - ascorbic-acid - vitamin-c - in-vitro - age - selenium - supplementation
Objective: Aging and oxidative stress may lead to enhanced cellular damage and programmed cell death. to study the association of intrinsic apoptosis with age and the effect of antioxidant supplementation on intrinsic and UV-induced apoptosis in children, young and elderly people. Methods: The study was a 2 months, double-blind, randomized trial. Three age groups were studied: children, young adults and elderly people. A total of 274 healthy subjects were allocated to a group supplemented with moderate amounts of retinol, beta-carotene, alpha-tocopherol, ascorbic acid and selenium or placebo. Plasma oxidative stress parameters were detected and apoptosis of lymphocytes was evaluated with TUNEL staining. Results: At baseline, percentages of intrinsic apoptosis were 13.8% and 11.1% in elderly and young people, respectively, both significantly higher than children (6.3%). A decrease of 1.7% and 2.3% in intrinsic apoptosis of lymphocytes was found in the supplemented groups of young and elderly people compared with their control groups (all p values
Physiological parameters of Bacillus cereus marking the end of acid-induced lag phases
Biesta-Peters, E.G. ; Mols, J.M. ; Reij, M.W. ; Abee, T. - \ 2011
International Journal of Food Microbiology 148 (2011)1. - ISSN 0168-1605 - p. 42 - 47.
flow-cytometry - bacterial-growth - intracellular ph - temperature - state
During lag phases microbial cells adapt to their environment and prepare to proliferate. Physiological parameters of B. cereus cells upon exposure to near-growth-boundary acid stress were investigated and markers for the transition between lag phase and growth were identified using fluorescent probes combined with flow cytometry. Determination of cell counts and optical density revealed lag phases of 1 h, 2 h and 5 h, in cultures shifted to pH 7, pH 5.3 (set with lactic acid) and pH 4.9 (set with sulfuric acid), respectively. The obtained lag phases fitted the trends in ATP levels, which were constant during the lag phase and increased after the onset of growth. Both the percentage of PI-stained cells and cells with a significant membrane potential decreased during the lag phase. This points to repair of membrane damage and the loss of membrane potential. However, both trends extended in the growth phase, thus not suitable to mark the onset of growth. The activity of the electron transfer chain and esterases did allow for assessment of transition between lag and growth phase. These activities were generally low during the lag phase and increased after the onset of growth. Our results show that, independent of the duration of the lag phase, for different conditions the same physiological trends could be observed. The change in signal of selected probes can be used as a marker for transition from lag phase to the growth phase and may aid in identification of novel targets interfering with bacterial exit from lag phase.
Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: Phenotype and transcriptome analysis
Melis, C.C.J. van; Nierop Groot, M.N. ; Tempelaars, M.H. ; Moezelaar, R. ; Abee, T. - \ 2011
Food Microbiology 28 (2011)2. - ISSN 0740-0020 - p. 275 - 283.
gram-positive bacteria - subtilis spores - vegetative cells - flow-cytometry - organic-acids - cold-shock - membrane - inhibition - genes - identification
Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of Bacillus cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth were assessed at pH 5.5 without and with 0.75, 1.5 and 3.0 mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from reduced germination and outgrowth rates to complete blockage of germination at 3.0 mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5 mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multidrug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed.
Alternative microbial methods: An overview and selection criteria.
Jasson, V. ; Jacxsens, L. ; Luning, P.A. ; Rajkovic, A. ; Uyttendaele, M. - \ 2010
Food Microbiology 27 (2010)6. - ISSN 0740-0020 - p. 710 - 730.
escherichia-coli o157-h7 - real-time pcr - latex agglutination tests - atp-bioluminescence method - in-situ hybridization - plate-count method - listeria-monocytogenes - rapid detection - raw-milk - flow-cytometry
This study provides an overview and criteria for the selection of a method, other than the reference method, for microbial analysis of foods. In a first part an overview of the general characteristics of rapid methods available, both for enumeration and detection, is given with reference to relevant bibliography. Perspectives on future development and the potential of the rapid method for routine application in food diagnostics are discussed. As various alternative “rapid” methods in different formats are available on the market, it can be very difficult for a food business operator or for a control authority to select the most appropriate method which fits its purpose. Validation of a method by a third party, according to international accepted protocol based upon ISO 16140, may increase the confidence in the performance of a method. A list of at the moment validated methods for enumeration of both utility indicators (aerobic plate count) and hygiene indicators (Enterobacteriaceae, Escherichia coli, coagulase positive Staphylococcus) as well as for detection of the four major pathogens (Salmonella spp., Listeria monocytogenes, E. coli O157 and Campylobacter spp.) is included with reference to relevant websites to check for updates. In a second part of this study, selection criteria are introduced to underpin the choice of the appropriate method(s) for a defined application. The selection criteria link the definition of the context in which the user of the method functions – and thus the prospective use of the microbial test results – with the technical information on the method and its operational requirements and sustainability. The selection criteria can help the end user of the method to obtain a systematic insight into all relevant factors to be taken into account for selection of a method for microbial analysis
Rapid drug susceptibility testing of mycobacteria by culture on a highly porous ceramic support
Ingham, C.J. ; Ayad, A.B. ; Nolsen, K. ; Mulder, B. - \ 2008
The International Journal of Tubercolosis and Lung Disease 12 (2008)6. - ISSN 1027-3719 - p. 645 - 650.
flow-cytometry - tuberculosis - assay - diagnosis - resistance - infection - anopore - growth - gene - tb
BACKGROUND: Phenotypic, culture-based methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis are relatively simple and may be particularly appropriate for resource-limited settings where tuberculosis (TB) is most prevalent. However, these methods can be slow and generate significant amounts of infectious waste. Low-cost digital imaging and a unique porous ceramic support for cell culture (Anopore) may offer opportunities to improve this situation. OBJECTIVE: To testa rapid DST method based on fluorescence microscopy of mycobacteria grown for a few generations on Anopore. DESIGN: Mycobacteria were cultured with and without drugs, and the resulting microcolonies were heat-killed and stained with the fluorogenic dye Syto16. Micros-copy, image-capture with a charge-coupled device camera and digital processing were used to quantify the inhibition of growth by drugs. Rapid DST for rifampicin and isoniazid was performed for clinical isolates. RESULTS: Mycobacteria could be cultured, killed, stained and imaged on Anopore. For DST, the Anopore method gave an accurate result in 3 days. CONCLUSION: This is an unprecedented speed for culture-based DST for this group of organisms and results in minimal infectious waste (<20 000 colony forming units). Analysis of mycobacteria by fluorescence and electron microscopy on Anopore also opens up research possibilities.
Ecophysiology of the developing total bacterial and Lactobacillus communities in the terminal small intestine of weaning piglets
Pieper, R. ; Janzcyk, P. ; Zeyner, A. ; Smidt, H. ; Guiard, V. ; Souffrant, W.B. - \ 2008
Microbial Ecology 56 (2008)3. - ISSN 0095-3628 - p. 474 - 483.
in-situ hybridization - targeted oligonucleotide probes - fecal samples - gastrointestinal microbiota - postnatal-development - porcine microbiota - escherichia-coli - flow-cytometry - immune-system - lactic-acid
Weaning of the pig is generally regarded as a stressful event which could lead to clinical implications because of the changes in the intestinal ecosystem. The functional properties of microbiota inhabiting the pig's small intestine (SI), including lactobacilli which are assumed to exert health-promoting properties, are yet poorly described. Thus, we determined the ecophysiology of bacterial groups and within genus Lactobacillus in the SI of weaning piglets and the impact of dietary changes. The SI contents of 20 piglets, 4 killed at weaning (only sow milk and no creep feed) and 4 killed at 1, 2, 5, and 11 days post weaning (pw; cereal-based diet) were examined for bacterial cell count and bacterial metabolites by fluorescence in situ hybridization (FISH). Lactobacilli were the predominant group in the SI except at 1 day pw because of a marked reduction in their number. On day 11 pw, bifidobacteria and E. coli were not detected, and Enterobacteriaceae and members of the Clostridium coccoides/Eubacterium rectale cluster were only found occasionally. L. sobrius/L. amylovorus became dominant species whereas the abundance of L. salivarius and L. gasseri/johnsonii declined. Concentration of lactic acid increased pw whereas pH, volatile fatty acids, and ammonia decreased. Carbohydrate utilization of 76 Lactobacillus spp. isolates was studied revealing a shift from lactose and galactose to starch, cellobiose, and xylose, suggesting that the bacteria colonizing the SI of piglets adapt to the newly introduced nutrients during the early weaning period. Identification of isolates based on partial 16S rRNA gene sequence data and comparison with fermentation data furthermore suggested adaptation processes below the species level. The results of our study will help to understand intestinal bacterial ecophysiology and to develop nutritional regimes to prevent or counteract complications during the weaning transition
A new whole-mount DNA quantification method and the analysis of nuclear DNA content in the stem-cell niche of Arabidopsis roots
Willemse, J. ; Kulikova, O. ; Jong, H. de; Bisseling, T. - \ 2008
The Plant Journal 55 (2008)5. - ISSN 0960-7412 - p. 886 - 894.
flow-cytometry - thaliana root - fluorescence - division - meristem - growth
A semi-automated method to quantify fluorescence intensity of objects in intact organs and tissues, composed of several cell layers, has been designed. The method has been developed on whole-mount propidium-iodide stained Arabidopsis thaliana (Arabidopsis) root tips, in which the DNA content of individual nuclei could be quantified. A diameter of less than 150 microm makes this organ most appropriate for whole-mount imaging. Further advantages are the lack of chlorophyll and transparent cell walls, with only a little background fluorescence. The method has a great advantage over flow cytometry, as the information regarding the positions of nuclei is maintained, and nuclei with aberrant DNA content can be re-assessed individually, which facilitates the efficient distinction between technical artefact and aberrant DNA content. Our averaging 3D method calculates the average of the summed fluorescence intensities of all sections of a nucleus and interpolates the missing sections, thereby allowing for the correction of detection problems. Furthermore, this method has the advantage of detecting objects in tissues covering multiple cell layers. The results of our method in Arabidopsis root tips showed that the quiescent centre cells, which rarely divide, are diploid, and are arrested in G1 or G0. Most stem cells, with the exception of those of the vascular tissue, are diploid cells, and their rather low division rate is caused by an elongated G1 phase. In contrast, the majority of the vascular stem cells are tetraploid.
S-antigen specific T helper type 1 response is present in Behcet's disease
Zhao, Changlin ; Yang, P. ; He, H. ; Lin, X. ; Li, B. ; Zhou, H. ; Huang, Xiangkun ; Kijlstra, A. - \ 2008
Molecular Vision 14 (2008). - ISSN 1090-0535 - p. 1456 - 1464.
cytokine production - flow-cytometry - cells - uveitis - autoimmunity - autoantigen - frequencies - diagnosis - elispot - il-23
PURPOSE: To investigate the frequency and phenotypic and functional characteristics of S-antigen (S-Ag) specific T cells in patients with Behcet's disease (BD). METHODS: Blood was taken from 23 active BD patients, 12 inactive BD patients, and 14 healthy controls. The clinical features of the patients were summarized. T cell response against 40 mixed S-Ag peptides was identified by interferon gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT). CD69 and CD45RO were used to characterize the phenotype of S-Ag specific T cells. The functional property of S-Ag specific T cells was investigated by measuring the production of cytokines. RESULTS: Response to the mixed S-Ag peptides was found in 56.5% and 25% of active and inactive BD patients, respectively. The responsiveness to S-Ag peptides was unrelated to the clinical features of the patients. About 65.8% of IFN-gamma(+) CD4(+) T cells in active BD patients expressed CD69 and CD45RO concomitantly. S-Ag peptides significantly induced a production of IFN-gamma and tumor necrosis factor (TNF)-alpha but not interleukin (IL)-2, IL-4, and IL-17 by peripheral blood mononuclear cells (PBMCs) in active BD patients with a response to S-Ag. CONCLUSIONS: S-Ag specific T cells are present in certain active BD patients, and most of them are activated memory CD4(+) T cells. These T cells may be involved in the pathogenesis of BD via producing Th1-dominant cytokines
Intestinal integrity and Akkermansia muciniphila: a mucin-degrading member of the intestinal microbiota present in infants, adults and elderly
Collado, M.C. ; Derrien, M.M.N. ; Isolauri, E. ; Vos, W.M. de; Salminen, S. - \ 2007
Applied and Environmental Microbiology 73 (2007)23. - ISSN 0099-2240 - p. 7767 - 7770.
16s ribosomal-rna - human fecal samples - flow-cytometry - diversity - communities - bacteria - gut - bifidobacteria - reveals - probes
Fluorescence in situ hybridization and real-time PCR analysis targeting the 16S rRNA gene of Akkermansia muciniphila were performed to determine its presence in the human intestinal tract. These techniques revealed that an A. muciniphila-like bacterium is a common member of the human intestinal tract and that its colonization starts in early life and develops within a year to a level close to that observed in adults (10(8) cells/g) but decreases (P <0.05) in the elderly.
On the way to cyanobacterial blooms: impact of the herbicide metribuzin on the competition between a green alga (Scenedesmus) and a cyanobacterium (Microcystis)
Lürling, M.F.L.L.W. ; Roessink, I. - \ 2006
Chemosphere 65 (2006)4. - ISSN 0045-6535 - p. 618 - 626.
fresh-water zooplankton - boreal forest lake - chlorophyll fluorescence - phytoplankton community - metsulfuron methyl - risk-assessment - flow-cytometry - daphnia-magna - hexazinone - toxicity
The hypothesis that exposure to a common and widely applied photosynthesis-inhibiting herbicide, metribuzin, would alter the outcome of the competitive battle between susceptible green algae (Scenedesmus obliquus) and tolerant cyanobacteria (Microcystis aeruginosa) was tested. In a long-term (17d) experiment, Scenedesmus and Microcystis populations as well as mixtures that started with different inoculum composition (i.e. 3:1, 1:1 and 1:3 Scenedesmus:Microcystis) were grown in the absence or presence of metribuzin (100mugl(-1)). In the absence of metribuzin, Scenedesmus was competitively superior and out-competed Microcystis regardless the initial composition of the mixed communities. However, this competitive outcome was reversed completely in the presence of metribuzin, where despite growth inhibition Microcystis became dominant. Hence, photosynthesis-inhibiting herbicides may not only affect algal community structure, but also provide cyanobacteria founder populations a window for dominance and thus play an important role in promoting cyanobacteria blooms.
Reduction of cell size induced by enod40 in Arabidopsis thaliana
Guzzo, F. ; Portaluppi, P. ; Grisi, R. ; Barone, S. ; Zampieri, S. ; Franssen, H. ; Levi, M. - \ 2005
Journal of Experimental Botany 56 (2005)412. - ISSN 0022-0957 - p. 507 - 513.
soybean nodule development - plant development - sucrose synthase - gene-expression - flow-cytometry - growth - division - cultures - rna - organogenesis
An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants
Colonic microbiota signatures across five northern European countries
Lay, C. ; Rigottier-Gois, L. ; Holmstrom, K. ; Rajilic-Stojanovic, M. ; Vaughan, E.E. ; Vos, W.M. de; Collins, M.D. ; Thiel, R. ; Namsolleck, P. ; Blaut, M. ; Dore, J. - \ 2005
Applied and Environmental Microbiology 71 (2005)7. - ISSN 0099-2240 - p. 4153 - 4155.
16s ribosomal-rna - in-situ hybridization - human fecal samples - targeted oligonucleotide probes - human feces - flow-cytometry - communities - bacteria - quantification - microflora
The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed
Design of a confocal microfluidic particle sorter using fluorescent photon burst detection
Kunst, B.H. ; Schots, A. ; Visser, A.J.W.G. - \ 2004
Review of Scientific Instruments 75 (2004)9. - ISSN 0034-6748 - p. 2892 - 2898.
single-molecule detection - correlation spectroscopy - submicrometer channels - cross-correlation - fluidic channels - flow-cytometry - cell sorter - dna - microcapillary - confinement
An instrumental system is described for detecting and sorting single fluorescent particles such as microspheres, bacteria, viruses, or even smaller macromolecules in a flowing liquid. The system consists of microfluidic chips (biochips), computer controlled high voltage power supplies, and a fluorescence microscope with confocal optics. The confocal observation volume and detection electro-optics allow measurements of single flowing fluorescent particles. The output of the avalanche photodiode (single photon detector) is coupled to a real-time photon-burst detection device, which output can address the control of high voltage power supplies for sorting purposes. Liquid propulsion systems like electro-osmotic flow and plain electric fields to direct the particles through the observation volume have been tested and evaluated. The detection and real-time sorting of fluorescent microspheres are demonstrated. Applications of these biochips for screening of bacteriophages-type biolibraries are briefly discussed. (C) 2004 American Institute of Physics.
Factors affecting survival of Clavibacter michiganesis subsp. sepedonicus in water
Wolf, J.M. van der; Beckhoven, J.R.C.M. van - \ 2004
Journal of Phytopathology 152 (2004)3. - ISSN 0931-1785 - p. 161 - 168.
bacterial ring rot - corynebacterium-sepedonicum - contaminated surfaces - nonculturable state - flow-cytometry - soil - resuscitation - starvation - resistance - viability
The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non-sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non-mucoid strain was also studied in sterile ditch water and simulated 'drainage water', in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non-culturable state