Records 1 - 20 / 272
Control mechanisms of microtubule overlap regions
Jongerius, Aniek - \ 2017
Wageningen University. Promotor(en): M.E. Janson. - Wageningen : Wageningen University - ISBN 9789463436113 - 133
microtubuli - celbiologie - plantencelbiologie - modellen - cellen - microtubules - cellular biology - plant cell biology - models - cells
Microtubule organization in cells is an important process. An example of careful microtubule organization is the mitotic spindle. The spindle is a bipolar structure with microtubules emanating from the poles at both sides. These microtubules form antiparallel overlaps in the centre of the spindle where they are bundled by bundling proteins. The overlaps are centred in the spindle and their constant length is regulated. The overlaps are important for the stability of the microtubule network, without bundling proteins the overlaps are lost and the spindle collapses. The antiparallel overlaps are also the site where microtubules slide apart to induce spindle elongation. Sliding is induced by tetrameric motor proteins that can bind to two bundled microtubules. Spindle elongation also requires microtubule growth at the overlaps. All these different functions at the overlap, sliding, growth/shrinkage and bundling, have to cooperate to maintain overlap length. While sliding reduces overlap length, growth will increase overlap length. These activities have to be coordinated for the maintenance of a constant overlap length. We propose that a feedback mechanism is present where growth of the microtubules is limiting the sliding in the overlap. This would prevent sliding when the overlap decreases and helps to maintain the overlap. We designed in vitro experiments to make antiparallel overlaps in vitro. In these experiments we use purified proteins from S. pombe. We combine ase1 and klp9 in a relative sliding assay to mimic the sliding in the midzone. In our experiments we combine relative sliding with dynamic microtubules for the first time. This allows us to test how these activities are coordinated. In other experiments we combine ase1 and cls1 with dynamic microtubules to see if the rescue activity of cls1 can be confined to the overlaps. Furthermore, interactions between motor proteins and diffusive proteins are investigated on single microtubules.
The fatter the better : selecting microalgae cells for outdoor lipid production
Dominguez Teles, I. - \ 2016
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Maria Barbosa; Dorinde Kleinegris. - Wageningen : Wageningen University - ISBN 9789462578821 - 164
algae - chlorococcum - lipids - lipogenesis - fat - production - phenotypes - inoculum - diameter - cells - sorting - algen - chlorococcum - lipiden - lipogenese - vet - productie - fenotypen - entstof - diameter - cellen - sorteren
In chapter 1 we introduce microalgae, photosynthetic microorganisms with potential to replace commodities (such as food, feed, chemicals and fuels). Production costs are still high, reason why microalgae are still only economically feasible for niche markets. We suggest to borrow the concept of plant domestication to select industrial microalgae cells. Two approaches can be successfully used to domesticate microalgae: adaptive laboratory evolution (ALE) and fluorescence assisted cell sorting (FACS). ALE takes advantage of the natural adaptability of microorganisms to different environments, while FACS actually select cells with specific phenotypes. This thesis aimed to select cells of Chlorococcum littorale with improved phenotypes, assuming that these cells could establish new populations with increased industrial performance.
In Chapter 2 we wanted to know what happened during time to biomass and lipid productivities of Chlorococcum littorale repeatedly subjected to N-starvation. We tested 2 different cycles of N-starvation, short (6 days) and long (12 days). Short cycles didn’t affect lipid productivity, highlighting the potential of C. littorale to be produced in semi-continuous cultivation. Repeated cycles of N-starvation could have caused adaptations of the strain. Hence, we also discussed the implications of using repeated N-starvation for adaptive laboratory evolution (ALE) experiments. Chapter 3 introduces a method to detect and to select microalgae cells with increased lipid content. The method requires only the fluorescence dye Bodipy505/515 dissolved in ethanol, and the method was designed to maintain cellular viability so the cells could be used to produce new inoculum. In chapter 4 we evaluated a question that emerged while deciding which criteria to use to sort lipid-rich cells: does cellular size affects lipid productivity of C. littorale? We hypothesized that cells with different diameters have different division rates, which could affect lipid productivity. Therefore, we assessed the influence of cell diameter, as a sorting parameter, on both biomass and lipid productivity of Chlorococcum littorale (comparing populations before and after sorting, based on different diameters). Results showed that the size of vegetative cells doesn’t affect the lipid productivity of C. littorale. In chapter 5 we present a strategy to sort cells of C. littorale with increased TAG productivity using the method developed at chapter 3. Both the original and the sorted population with the highest lipid productivity (namely, S5) were compared under simulated Dutch summer conditions. The results confirmed our data from experiments done under continuous light: S5 showed a double TAG productivity. Our results showed also that the selected phenotype was stable (1.5 year after sorting) and with potential to be used under industrial conditions. In chapter 6 we extrapolated our results (indoor and outdoor) to other climate conditions. We ran simulations changing the light conditions to four different locations worldwide (the Netherlands, Norway, Brazil and Spain) to estimate both biomass and TAG productivities. Results indicated that biomass yields were reduced at locations with higher light intensities (Brazil/Spain) when compared with locations with lower light intensities (Norway/Netherlands). Hence, the choice of location should not be based on light intensity, but on how stable irradiation is. Chapter 7 is the general discussion of the thesis, demonstrating that both ALE and FACS are effective approaches to select industrial microalgae cells. We also present our view on how ALE and FACS could further improve microalgae strains for industry.
Replacing animal experiments in developmental toxicity testing of phenols by combining in vitro assays with physiologically based kinetic (PBK) modelling
Strikwold, Marije - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Ruud Woutersen, co-promotor(en): Ans Punt. - Wageningen : Wageningen University - ISBN 9789462576926 - 169
animal experiments - animal testing alternatives - toxicity - testing - phenols - in vitro - embryonic stem cells - tissues - cells - dosage - toxicology - animal health - dierproeven - alternatieven voor dierproeven - toxiciteit - testen - fenolen - in vitro - embryonale stamcellen - weefsels - cellen - dosering - toxicologie - diergezondheid
Waves of change: immunomodulation of the innate immune response by low frequency electromagnetic field exposure
Golbach, L.A. - \ 2015
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Lidy van Kemenade. - Wageningen : Wageningen University - ISBN 9789462574090 - 172
immuniteitsreactie - elektromagnetisch veld - cellen - celbiologie - blootstelling - immune response - electromagnetic field - cells - cellular biology - exposure
In this thesis we investigated possible modulatory roles of low frequency electromagnetic fields (LF EMFs) exposure on the innate immune system. Recent decades have seen a huge increase in the use of electronic devices that nowadays enable us to communicate with distant family, enjoy music everywhere or order food without leaving the house. However besides the benefits, this evolution has also resulted in increased public concern about the potential adverse health effects of non-ionizing radiation. Every power line or electronic device emits a wide range of electromagnetic waves, which can pass through our bodies or damage our skin, depending on the characteristics of the waves. The symptoms attributed to continuous EMF exposure range from non-specific physical symptoms, such as fatigue 1, headaches 2, and redness of the skin to increased prevalence of childhood leukaemia 3. Although many theories regarding a potential mechanism of induction are put forward, to date no clear mechanism of action has been elucidated. Experimental evidence that could support an association between exposure and health status appears to be insufficient and inconsistent 4,5. We investigated the potential effect of LF EMF exposure on neutrophils, one of the key players of the innate immune response. We tried to elucidate a possible mechanism of interaction between intracellular signalling pathways and LF EMF exposure. We aimed to investigate calcium signalling, actin reorganization, cell migration and antimicrobial activity during exposure with different in vitro approaches.
Genetic and Non-Genetic Inheritance of Natural Antibodies Binding Keyhole Limpet Hemocyanin in a Purebred Layer Chicken Line
Berghof, T.V.L. ; Klein, S.A.S. van der; Arts, J.A.J. ; Parmentier, H.K. ; Poel, J.J. van der; Bovenhuis, H. - \ 2015
PLoS ONE 10 (2015)6. - ISSN 1932-6203 - 13 p.
laying hens - immune-responses - parameters - iga - survival - isotypes - associations - sensitivity - disease - cells
Natural antibodies (NAb) are defined as antibodies present in individuals without known antigenic challenge. Levels of NAb binding keyhole limpet hemocyanin (KLH) in chickens were earlier shown to be heritable, and to be associated with survival. Selective breeding may thus provide a strategy to improve natural disease resistance. We phenotyped 3,689 white purebred laying chickens for KLH binding NAb of different isotypes around 16 weeks of age. Heritabilities of 0.12 for the titers of total antibodies (IgT), 0.14 for IgM, 0.10 for IgA, and 0.07 for IgG were estimated. We also estimated high, positive genetic, and moderate to high, positive phenotypic correlations of IgT, IgM, IgA, and IgG, suggesting that selective breeding for NAb can be done on all antibody isotypes simultaneously. In addition, a relatively substantial non-genetic maternal environmental effect of 0.06 was detected for IgM, which may reflect a transgenerational effect. This suggests that not only the genes of the mother, but also the maternal environment affects the immune system of the offspring. Breaking strength and early eggshell whiteness of the mother’s eggs were predictive for IgM levels in the offspring, and partly explained the observed maternal environmental effects. The present results confirm that NAb are heritable, however maternal effects should be taken into account.
Quercetin tests negative for genotoxicity in transcriptome analyses of liver and small intestine of mice
Hil, E.F. van den; Schothorst, E.M. van; Stelt, I. van der; Keijer, J. ; Rietjens, I. - \ 2015
Food and Chemical Toxicology 81 (2015). - ISSN 0278-6915 - p. 34 - 39.
in-vivo - reverse mutation - bone-marrow - dna-damage - flavonoids - rats - cells - carcinogenicity - mutagenicity - polyphenols
Given the positive results of quercetin in in vitro genotoxicity studies, the in vivo genotoxic properties of this important dietary flavonoid warrant testing, especially considering possible high intake via widely available food supplements. Here, this was done by transcriptome analyses of the most relevant tissues, liver and small intestine, of quercetin supplemented mice. Quercetin (0.33%) supplemented to a high-fat diet was administered to mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. Microarray pathway analysis of liver and small intestine showed no regulation of genotoxicity related pathways. Analysis of DNA damage related genes also did not point at genotoxicity. Furthermore, a published classifier set of transcripts for identifying genotoxic compounds did not indicate genotoxicity. Only two transcripts of the classifier set were regulated, but in the opposite direction compared with the genotoxic compounds 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1). Based on the weight of evidence of three different types of analysis, we conclude that supplementation with quercetin at ~350¿mg/kg bw/day for 12 weeks in mice showed no up-regulation of genotoxicity related pathways in liver and small intestine.
Injectable nanoparticle-loaded hydrogen system for local delivery of sodium alendronate
Posadowska, U. ; Parizek, M. ; Filova, E. ; Wlodarczyk-Biegun, M.K. ; Kamperman, M.M.G. ; Bacakova, L. ; Pamula, E. - \ 2015
International Journal of Pharmaceutics 485 (2015)1. - ISSN 0378-5173 - p. 31 - 40.
drug-delivery - osteoclast formation - controlled-release - bone - gellan - cytotoxicity - formulation - cells - microspheres - therapeutics
Systemic administration of bisphosphonates, e.g. sodium alendronate (Aln) is characterized by extremely low bioavailability and high toxicity. To omit aforementioned drawbacks an injectable system for the intra-bone delivery of Aln based on Aln-loaded nanoparticles (NPs-Aln) suspended in a hydrogel matrix (gellan gum, GG) was developed. Aln was encapsulated in poly(lactide-co-glycolide) (PLGA 85:15) by solid–oil–water emulsification. Drug release tests showed that within 25 days all the encapsulated drug was released from NPs-Aln and the release rate was highest at the beginning and decreased with time. In contrast, by suspending NPs-Aln in a GG matrix, the release rate was significantly lower and more constant in time. The GG–NPs-Aln system was engineered to be easily injectable and was able to reassemble its structure after extrusion as shown by rheological measurements. Invitro studies showed that the GG–NPs-Aln was cytocompatible with MG-63 osteoblast-like cells and it inhibited RANKL-mediated osteoclastic differentiation of RAW 264.7 cells. The injectability, the sustained local delivery of small doses of Aln and the biological activity render the GG–NPs-Aln system promising for the local treatment of osteoporosis and other bone tissue disorders.
First clinical results of a personalized immunotherapeutic vaccine against recurrent, incompletely resected, treatment-resistant glioblastoma multiforme (GBM) tumors, based on combined all- and auto-immune tumor reactivity
Schijns, V.E.J.C. ; Pretto, C. ; Devillers, L. ; Pierre, D. ; Hofman, F.M. ; Chen, T.C. ; Mespouille, P. ; Hantos, P. ; Glorieux, P. ; Bota, D.A. ; Stathopolous, A. - \ 2015
Vaccine 33 (2015)23. - ISSN 0264-410X - p. 2690 - 2696.
colony-stimulating factor - survival - melanoma - cells - cyclophosphamide - adjuvant - immunity - glioma
Glioblastoma multiforme (GBM) patients have a poor prognosis. After tumor recurrence statistics suggestan imminent death within 1–4.5 months. Supportive preclinical data, from a rat model, provided therational for a prototype clinical vaccine preparation, named Gliovac (or ERC 1671) composed of autologousantigens, derived from the patient’s surgically removed tumor tissue, which is administered together withallogeneic antigens from glioma tissue resected from other GBM patients. We now report the first resultsof the Gliovac treatment for treatment-resistant GBM patients.Nine (9) recurrent GBM patients, after standard of care treatment, including surgery radio- andchemotherapy temozolomide, and for US patients, also bevacizumab (AvastinTM), were treated under acompassionate use/hospital exemption protocol. Gliovac was given intradermally, together with humanGM-CSF (Leukine®), and preceded by a regimen of regulatory T cell-depleting, low-dose cyclophos-phamide.Gliovac administration in patients that have failed standard of care therapies showed minimal toxicityand enhanced overall survival (OS). Six-month (26 weeks) survival for the nine Gliovac patients was 100%versus 33% in control group. At week 40, the published overall survival was 10% if recurrent, reoperatedpatients were not treated. In the Gliovac treated group, the survival at 40 weeks was 77%. Our datasuggest that Gliovac has low toxicity and a promising efficacy. A phase II trial has recently been initiatedin recurrent, bevacizumab naïve GBM patients (NCT01903330).
Low-frequency electromagnetic field exposure enhances extracellular trap formation by human neutrophils through the NADPH pathway
Golbach, L.A. ; Scheer, M.H. ; Cuppen, J.J.M. ; Savelkoul, H.F.J. ; Verburg-van Kemenade, B.M.L. - \ 2015
Journal of Innate Immunity 7 (2015)5. - ISSN 1662-811X - p. 459 - 465.
magnetic-fields - release - macrophages - cells - myeloperoxidase - proliferation - monocytes - immunity - oxidase
Low-frequency (LF) electromagnetic fields (EMFs) are abundantly present in modern society, and the potential biological consequences of exposure to these fields are under intense debate. Immune cells are suggested as possible target cells, though a clear mechanism is lacking. Considering their crucial role in innate immune activation, we selected an ex vivo exposure set-up with human neutrophils to investigate a possible correlation between neutrophil extracellular trap (NET) formation and LF EMF exposure. Our study shows that formation of NETs is enhanced by LF EMF exposure. Enhanced NET formation leads to increased antimicrobial properties as well as damage to surrounding cells. We found that LF-EMF-induced NET formation is dependent on the NADPH oxidase pathway and production of reactive oxygen species. Additionally, LF EMF exposure does not influence autophagy and PAD4 activity. Our study provides a mechanism by which exposure to LF EMFs could influence the innate immune system.
2-Amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazin-3-one as an unexpected product from reduction of 5-methyl-2-nitrophenol
Jansze, S.M. ; Saggiomo, V. ; Marcelis, A.T.M. ; Lutz, M. ; Velders, A.H. - \ 2015
Tetrahedron Letters 56 (2015)9. - ISSN 0040-4039 - p. 1060 - 1062.
aromatic nitro-compounds - phenoxazinone synthase - azo-compounds - aminophenol - mechanism - cells
When attempting to synthesize symmetric 2,2'-dihydroxy-4,4'-dimethyl-azobenzene from 5-methyl-2-nitrophenol by reductive methods based on two literature procedures, an unexpected product was isolated in 30% yield. Full analysis by mass spectrometry, NMR spectroscopy, and single-crystal X-ray structure analysis, proved this product to be tricyclic 2-amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazin-3-one. This Letter conveys a warning regarding reductive synthetic routes toward azobenzenes. We also present a novel reductive synthetic route for phenoxazines, an important class of tricyclic compounds.
In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing
Nicolas, J.A.Y. ; Hendriksen, P.J.M. ; Haan, L.H.J. de; Koning, R. ; Rietjens, I.M.C.M. ; Bovee, T.F.H. - \ 2015
Toxicology in Vitro 29 (2015)2. - ISSN 0887-2333 - p. 281 - 288.
gated sodium-channels - membrane currents - calcium-channel - open-state - tetrodotoxin - toxins - cells - na+ - diphenhydramine - inhibition
The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na+, K+, or Ca2+ channels or the Na+/K+ ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K+ channel blockers, amiodarone, clofilium and sematilide, and the Na+/K+ ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na+ and Ca2+ channels, but less sensitive for the compounds affecting K+ channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins
Production in a factory (the cell) requires high level of organisation : the cell: The plant’s smallest building block
Heuvelink, E. - \ 2015
In Greenhouses : the international magazine for greenhouse growers 4 (2015)2. - ISSN 2215-0633 - p. 18 - 19.
glastuinbouw - plantenfysiologie - plantkunde - celbiologie - cellen - celstructuur - beschrijvingen - greenhouse horticulture - plant physiology - botany - cellular biology - cells - cell structure - descriptions
The cell is the plant’s smallest building block. Many cultivation techniques and climate control measures have an effect at this level. Some knowledge about the functioning of the cell is therefore very useful. Many components of the cell have bizarre names so to understand it all better, for the purpose of this article, we compare it to a factory.
BABY BOOM-induced somatic embryogenesis in Arabidopsis
Horstman, A. - \ 2015
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kim Boutilier. - Wageningen : Wageningen University - ISBN 9789462572317 - 233
arabidopsis - somatische embryogenese - in vitro kweek - cellen - weefselkweek - plantengroeiregulatoren - somatische embryo's - transcriptie - arabidopsis - somatic embryogenesis - in vitro culture - cells - tissue culture - plant growth regulators - somatic embryos - transcription
Under appropriate tissue culture conditions, somatic plant cells can be induced to form embryos in a process called somatic embryogenesis (SE). SE provides a way to clonally propagate desirable plants and is therefore an important plant breeding tool. SE has also fascinated scientists for decades as an expression of plant ‘totipotency’, the ability to regenerate a whole new individual through embryogenesis. This thesis aims to obtain a deeper understanding of somatic embryo induction in Arabidopsis by the transcription factor BABY BOOM (BBM), through identification and functional analysis of BBM-binding proteins and BBM target genes.
Chapter 1 introduces the concept of somatic embryogenesis, describes the different SE systems in Arabidopsis, and discusses the role of the plant hormone auxin and chromatin modifying proteins in this process. An overview is presented on the current knowledge on SE-induction through ectopic overexpression of certain transcription factor genes. These include BBM, as well as other genes that are studied in this thesis in relation to BBM.
BBM is part of the eight member AIL subfamily of AP2/ERF domain transcription factors. Chapter 2 reviews the role of AIL proteins during embryogenesis, stem cell niche specification, meristem maintenance and organ positioning and growth. We summarize the gene regulatory networks in which AILs function and describe how these transcription factors integrate multiple hormonal inputs, with special emphasis on the interactions between AILs and auxin. Finally, we conclude that although the functions of AILs in plant development are well described, knowledge on the molecular mode of action of AIL proteins and the identity of AIL target genes is still limited.
Transcription factors function in protein complexes and in Chapter 3 we show that members of the HOMEODOMAIN GLABROUS (HDG) transcription factor family physically interact with BBM and other AILs. HDG genes are expressed in the epidermis, the outer cell layer of the plant, where they promote differentiation of cells into specialized epidermal cell types, such as trichomes or stomata. We show that ectopic overexpression of HDG1 leads to loss of root and shoot meristems, phenotypes that had previously been reported for loss-of-function ail mutants. Conversely, down-regulation of HDG genes led to reduced cell differentiation, enhanced cell proliferation and SE phenotypes, phenotypes that resemble those found in AIL overexpression lines. Moreover, we found that co-overexpression of BBM and HDG1 reduces the overexpression phenotypes of both proteins. These results suggest opposite functions of AIL and HDG transcription factors, with AILs stimulating cell proliferation and HDGs stimulating cell differentiation, with the ratio between the two proteins determining the developmental outcome. Finally, we show that HDGs and AILs regulate each other on a transcriptional level and that they share common target genes.
A variety of AIL overexpression phenotypes has been described in the literature, with BBM and PLT5/AIL5 being the only known AILs that induce SE upon overexpression. We show in Chapter 4 that all AIL proteins except AIL1 and ANT are able to induce SE, but that this phenotype relies on a high AIL protein dosage. Using BBM and PLT2 as AIL representatives, we show that an intermediate AIL concentration induces organogenesis (ectopic root and shoot formation) and that a low concentration inhibits cellular differentiation. In addition, we show that BBM and PLT2 induce direct SE when activated at seed germination, while post-germination activation leads to indirect SE from callus. The LEAFY COTYLEDON (LEC)/LAFL genes, which also encode SE-inducing transcription factors, are direct targets of BBM/PLT2 during direct SE, showing that these two SE pathways are linked. Using LAFL gene mutants, we show that the LAFL pathway is an important downstream component of BBM-mediated SE.
Chapter 5 presents the in vivo, genome-wide analysis of BBM DNA binding sites in somatic embryos using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our ChIP-seq and gene expression analysis reveal that BBM binds and positively regulates auxin biosynthesis genes and the recently discovered positive regulators of SE, the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes. Knock-out of either pathway reduced BBM-mediated SE, showing that auxin biosynthesis and the AHL genes are important components of the BBM pathway. We also show that BBM binds to a consensus DNA motif that resembles the reported ANT binding motif.
Chapter 6 reviews methods for identifying the direct target genes of a plant transcription factor using microarrays, as was done for HDG1 (Chapter 4). We describe which different systems can be used to control transcription factor activity, and how these can be combined with microarray analysis to identify target genes. In addition, we provide guidelines for the statistical analysis of microarray data and for the confirmation of candidate target genes.
In plant biology, protein-protein interactions are often studied using bimolecular fluorescence complementation (BiFC) or split-YFP. In my BBM-HDG interaction studies I encountered problems using this method, which lead to the cautionary note on the use of BiFC presented in Chapter 7. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. However, because the fluorescent protein halves are prone to self-assembly, it is crucial to use proper controls and a quantitative read-out of fluorescence to avoid false positive interactions. We present a guideline for the setup of a BiFC experiment, discussing each step in the protocol.
Chapter 8 discusses how the results presented in this thesis contribute to our knowledge on AIL transcription factors and somatic embryo induction, as well as the questions that still remain. An extended model of dose-dependent AIL function is proposed, as well as mechanisms by which the AIL-HDG interaction could function at the molecular level. Finally, an overview is provided of the molecular-genetic intersection between the different transcription factor-induced SE pathways.
Partitioning of humic acids between aqueous solution and hydrogel. 3. Microelectrodic dynamic speciation analysis of free and bound humic metal complexes in the gel phase
Yasadi, K. ; Pinheiro, J.P. ; Zielinska, K. ; Town, R.M. ; Leeuwen, H.P. van - \ 2015
Langmuir 31 (2015)5. - ISSN 0743-7463 - p. 1737 - 1745.
dissolved organic-matter - stability-constants - alginate gel - thin-films - adsorption - ions - fluorescence - substances - cells - soil
The hydrogel/water partitioning of the various species in the cadmium(II)/soil humic acid (HA) system is studied for two types of gel, using in situ microelectrodic voltammetry. Under the conditions of this work, with HA particles of ca. 25 and 125 nm radius, the CdHA complex is shown to be close to nonlabile toward a 12.5 µm radius microelectrode. This implies that its kinetic contribution to Cd2+ reduction at the medium/microelectrode interface is practically negligible. The polyacrylamide (PAAm) gels equilibrate with the aqueous medium under significant sorption of HA at the gel backbone/gel medium interface, which in turn leads to induced sorption of Cd(II) in the form of immobilized gel-bound CdHA. The rather high total Cd content of the PAAm gel suggests that the binding of Cd2+ by the hydrophobically gel-bound HA is stronger than that for dispersed HA particles. Still, the intraparticulate speciation of Cd(II) over Cd2+ and CdHA corresponds to an intrinsic stability constant similar to that for simple monocarboxylate ligands such as acetate. Alginate gels are negatively charged, and their free [Cdaq2+] is higher than that in the medium by the corresponding Donnan coefficient. On top of that, Cd2+ is specifically sorbed by the gel backbone/gel medium interface to reach accumulation factors as high as a few tens. HA and CdHA accumulate in the outer 20 µm film of gel at the gel/water interface of both gels, but they do not penetrate into the bulk of the alginate gel. Overall, the gel/water interface dictates drastic changes in the speciation of Cd/HA as compared to the aqueous medium, with distinct features for each individual type of gel. The results have broad significance, for example, for predictions of reactivity and bioavailability of metal species which inherently involve partitioning and diffusion into diverse gel layers such as biointerfacial cell walls, biofilm matrices, and mucous membranes.
Tracing the molecular basis of transcriptional dynamics in noisy data by using an experiment based mathematical model
Rybakova, K.N. ; Tomaszewska, A. ; Mourik, S. van; Blom, Joke ; Westerhoff, H.V. ; Carlberg, C. ; Bruggeman, F.J. - \ 2015
Nucleic acids research 43 (2015)1. - ISSN 0305-1048 - p. 153 - 161.
differentiation-related protein - gene-expression - parameter-estimation - rna degradation - adrp expression - receptor - cells - elongation - promoter - networks
Changes in transcription factor levels, epigenetic status, splicing kinetics and mRNA degradation can each contribute to changes in the mRNA dynamics of a gene. We present a novel method to identify which of these processes is changed in cells in response to external signals or as a result of a diseased state. The method employs a mathematical model, for which the kinetics of gene regulation, splicing, elongation and mRNA degradation were estimated from experimental data of transcriptional dynamics. The time-dependent dynamics of several species of adipose differentiation-related protein (ADRP) mRNA were measured in response to ligand activation of the transcription factor peroxisome proliferator-activated receptor d (PPARd). We validated the method by monitoring the mRNA dynamics upon gene activation in the presence of a splicing inhibitor. Our mathematical model correctly identifies splicing as the inhibitor target, despite the noise in the data.
Activation of the chicken type I IFN response by infectious bronchitis coronavirus
Kint, J. ; Fernandez Gutierrez, M.M. ; Maier, H.J. ; Britton, P. ; Langereis, M.A. ; Koumans, J. ; Wiegertjes, G.F. ; Forlenza, M. - \ 2015
Journal of Virology 89 (2015)2. - ISSN 0022-538X - p. 1156 - 1167.
mouse hepatitis-virus - singly labeled probes - double-stranded-rna - rig-i - murine coronavirus - innate immunity - receptor activation - viral-rna - cells - induction
Coronaviruses from both the Alpha and Betacoronavirus genera, interfere with the type I interferon (IFN) response in various ways, ensuring limited activation of the IFN response in most cell types. Of Gammacoronaviruses that mainly infect birds, little is known about activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells and in a chicken cell line. Ifnß expression in fact, is delayed with respect to the peak of viral replication and accompanying accumulation of dsRNA. In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the IFN response at the transcriptional and translational level. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, signalling of non-self dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.
Human buccal epithelium acquires microbial hyporesponsiveness at birth, a role for secretory leukocyte protease inhibitor
Menckeberg, C.L. ; Hol, J. ; Simons-Oosterhuis, Y. ; Raatgeep, H.R. ; Ruiter, L.F. de; Lindenbergh-Kortleve, D.J. ; Korteland-van Male, A.M. ; Aidy, S.F. El; Lierop, P.P.E. van; Kleerebezem, M. ; Groeneweg, M. ; Kraal, G. ; Elink-Schuurman, B.E. ; Jongste, J.C. de; Nieuwenhuis, E.E.S. ; Samsom, J.N. - \ 2015
Gut 64 (2015). - ISSN 0017-5749 - p. 884 - 893.
toll-like receptors - negative regulation - intestinal-mucosa - crohn-disease - responses - cells - tolerance - lipopolysaccharide - inflammation - mechanisms
Objective Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acquisition of hyporesponsiveness to microbial stimulation using primary human buccal epithelium. Design Buccal ECs collected directly after birth and in later stages of life were investigated. Chemokine release and regulatory signalling pathways were studied using primary buccal ECs and the buccal EC line TR146. Findings were extended to the intestinal mucosa using murine model systems. Results Directly after birth, primary human buccal ECs spontaneously produced the chemokine CXCL-8 and were responsive to microbial stimuli. Within the first weeks of life, these ECs attained hyporesponsiveness, associated with inactivation of the NF-¿B pathway and upregulation of the novel NF-¿B inhibitor SLPI but no other known NF-¿B inhibitors. SLPI protein was abundant in the cytoplasm and the nucleus of hyporesponsive buccal ECs. Knock-down of SLPI in TR146-buccal ECs induced loss of hyporesponsiveness with increased NF-¿B activation and subsequent chemokine release. This regulatory mechanism extended to the intestine, as colonisation of germfree mice elicited SLPI expression in small intestine and colon. Moreover, SLPI-deficient mice had increased chemokine expression in small intestinal and colonic ECs. Conclusions We identify SLPI as a new player in acquisition of microbial hyporesponsiveness by buccal and intestinal epithelium in the first weeks after microbial colonisation.
The small intestine microbiota, nutritional modulation and relevance for health
Aidy, S.F. El; Bogert, B. van den; Kleerebezem, M. - \ 2015
Current Opinion in Biotechnology 32 (2015). - ISSN 0958-1669 - p. 14 - 20.
human gut microbiota - y gastric bypass - bariatric surgery - host - disease - inflammation - diversity - responses - benefits - cells
The intestinal microbiota plays a profound role in human health and extensive research has been dedicated to identify microbiota aberrations that are associated with disease. Most of this work has been targeting the large intestine and fecal microbiota, while the small intestine microbiota may also have a profound impact on various aspects of the host's physiology, including immune, metabolic and endocrine functions. This review highlights the recent advances made in the study of the human small intestine microbiota. In addition, it describes recent human and animal studies that underpin the importance of this part of the intestine for health of the host organism.
Temporal proteomic analysis and label-free quantitation of viral proteins of an invertebrate iridovirus
Ince, I.A. ; Boeren, S. ; Oers, M.M. van; Vlak, J.M. - \ 2015
Journal of General Virology 96 (2015)1. - ISSN 0022-1317 - p. 196 - 205.
chilo-iridescent-virus - polypeptides - identification - type-6 - cells - membrane - genome - civ
Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a 212 kb-long linear double-stranded DNA genome that encodes 215 putative open reading frames. The IIV-6 virion-associated proteins consist of at least 54 virally-encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicates that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantitation using a proteomic approach. A total of 95 viral encoded proteins were detected in infected cells, of which 37 are virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles. These clusters were: 1) proteins with stably low or 2) exponentially increasing expression levels during infection, and 3) proteins that were initially highly abundant, but showed slightly reduced levels after 48 hours (h) post infection (p.i.). Here, we provide novel information on the kinetics of virion and infected cell-specific protein levels that assists in understanding gene regulation in this lesser known DNA virus model.
Staphylococcus aureus ST398 gene expression profiling during ex vivo colonization of porcine nasal epithelium
Tulinski, P. ; Duim, B. ; Wittink, F.R. ; Jonker, M.J. ; Breit, T.M. ; Putten, J.P. van; Wagenaar, J.A. ; Fluit, A.C. - \ 2014
BMC Genomics 15 (2014). - ISSN 1471-2164
clumping factor-b - methicillin-resistant - carriage - model - adherence - humans - proteinases - determinant - infections - cells
Background: Staphylococcus aureus is a common human and animal opportunistic pathogen. In humans nasal carriage of S. aureus is a risk factor for various infections. Methicillin-resistant S. aureus ST398 is highly prevalent in pigs in Europe and North America. The mechanism of successful pig colonization by MRSA ST398 is poorly understood. Previously, we developed a nasal colonization model of porcine nasal mucosa explants to identify molecular traits involved in nasal MRSA colonization of pigs. Results: We report the analysis of changes in the transcription of MRSA ST398 strain S0462 during colonization on the explant epithelium. Major regulated genes were encoding metabolic processes and regulation of these genes may represent metabolic adaptation to nasal mucosa explants. Colonization was not accompanied by significant changes in transcripts of the main virulence associated genes or known human colonization factors. Here, we documented regulation of two genes which have potential influence on S. aureus colonization; cysteine extracellular proteinase (scpA) and von Willebrand factor-binding protein (vWbp, encoded on SaPIbov5). Colonization with isogenic-deletion strains (Delta vwbp and Delta scpA) did not alter the ex vivo nasal S. aureus colonization compared to wild type. Conclusions: Our results suggest that nasal colonization with MRSA ST398 is a complex event that is accompanied with changes in bacterial gene expression regulation and metabolic adaptation.