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Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle
Patyshakuliyeva, A. ; Post, H. ; Zhou, M. ; Jurak, E. ; Heck, A.J.R. ; Hilden, K.S. ; Kabel, M.A. ; Makela, M.R. ; Altenaar, M.A.F. ; Vries, R.P. de - \ 2015
Environmental Microbiology 17 (2015)8. - ISSN 1462-2912 - p. 3098 - 3109.
fungus phanerochaete-chrysosporium - ceriporiopsis-subvermispora - mannitol dehydrogenase - fomitopsis-palustris - gene-expression - button mushroom - rot fungi - acid - lignocellulose - insights
The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and con-tributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exoproteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush
The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions
Karimi Jashni, M. ; Mehrabi, R. ; Collemare, J. ; Mesarich, C.H. ; Wit, P.J.G.M. de - \ 2015
Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 7 p.
cf-2-dependent disease resistance - extracellular serine-protease - l. enhances resistance - class iv chitinases - phytophthora-infestans - cladosporium-fulvum - proteolytic-enzymes - antifungal activity - gene-expression - tomato
Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.
Cell kinetics during regeneration in the sponge Halisarca caerulea: how local is the response to tissue damage?
Alexander, B.E. ; Achlatis, M. ; Osinga, R. ; Geest, H.G. van der; Cleutjens, J.P.M. ; Schutte, B. ; Goeij, J.M. de - \ 2015
PeerJ 3 (2015). - ISSN 2167-8359
coral-reef sponges - morphological strategies - sublittoral demosponge - chemical defenses - cycle checkpoints - gene-expression - life-history - trade-off - growth - population
Sponges have a remarkable capacity to rapidly regenerate in response to wound infliction. In addition, sponges rapidly renew their filter systems (choanocytes) to maintain a healthy population of cells. This study describes the cell kinetics of choanocytes in the encrusting reef sponge Halisarca caerulea during early regeneration (0–8 h) following experimental wound infliction. Subsequently, we investigated the spatial relationship between regeneration and cell proliferation over a six-day period directly adjacent to the wound, 1 cm, and 3 cm from the wound. Cell proliferation was determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h). The percentage of proliferative choanocytes increased over time in all areas and after six days of regeneration choanocyte proliferation rates were comparable to steady-state tissue. Tissue areas farther from the wound had higher rates of choanocyte proliferation than areas closer to the wound, indicating that more resources are demanded from tissue in the immediate vicinity of the wound. There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges. Our data suggest that the production of collagen-rich wound tissue is a key process in tissue regeneration for H. caerulea, and helps to rapidly occupy the bare substratum exposed by the wound. Regeneration and choanocyte renewal are competing and negatively correlated life-history traits, both essential to the survival of sponges. The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.
Organizer-Derived WOX5 Signal Maintains Root Columella Stem Cells through Chromatin-Mediated Repression of CDF4 Expression.
Pi, L. ; Graaff, E. van der; Llavata Peris, C.I. ; Weijers, D. ; Henning, L. ; Groot, E. de; Laux, T. - \ 2015
Developmental cell 33 (2015)5. - ISSN 1534-5807 - p. 576 - 588.
histone deacetylase - arabidopsis-thaliana - transcriptional repression - gene-expression - wuschel - meristem - shoot - topless - protein - fate
Stem cells in plants and animals are maintained pluripotent by signals from adjacent niche cells. In plants, WUSCHEL HOMEOBOX (WOX) transcription factors are central regulators of stem cell maintenance in different meristem types, yet their molecular mode of action has remained elusive. Here we show that in the Arabidopsis root meristem, the WOX5 protein moves from the root niche organizer, the quiescent center, into the columella stem cells, where it directly represses the transcription factor gene CDF4. This creates a gradient of CDF4 transcription, which promotes differentiation opposite to the WOX5 gradient, allowing stem cell daughter cells to exit the stem cell state. We further show that WOX5 represses CDF4 transcription by recruiting TPL/TPR co-repressors and the histone deacetylase HDA19, which consequently induces histone deacetylation at the CDF4 regulatory region. Our results show that chromatin-mediated repression of differentiation programs is a common strategy in plant and animal stem cell niches.
Parasitism overrides herbivore identity allowing hyperparasitoids to locate their parasitoid host using herbivore-induced plant volatiles
Zhu, F. ; Broekgaarden, C. ; Weldegergis, B.T. ; Harvey, J.A. ; Vosman, B. ; Dicke, M. ; Poelman, E.H. - \ 2015
Molecular Ecology 24 (2015)1. - ISSN 0962-1083 - p. 2886 - 2899.
cabbage brassica-oleracea - time rt-pcr - nicotiana-attenuata - insect herbivores - gene-expression - trophic levels - defense - responses - specialist - generalist
Foraging success of predators profoundly depends on reliable and detectable cues indicating the presence of their often inconspicuous prey. Carnivorous insects rely on chemical cues to optimize foraging efficiency. Hyperparasitoids that lay their eggs in the larvae or pupae of parasitic wasps may find their parasitoid hosts developing in different herbivores. They can use herbivore-induced plant volatiles (HIPVs) to locate parasitized caterpillars. Because different herbivore species induce different HIPV emission from plants, hyperparasitoids may have to deal with large variation in volatile information that indicates host presence. In this study, we used an ecogenomics approach to first address whether parasitized caterpillars of two herbivore species (Pieris rapae and P. brassicae) induce similar transcriptional and metabolomic responses in wild Brassica oleracea plants and, second, whether hyperparasitoids Lysibia nana are able to discriminate between these induced plant responses to locate their parasitoid host in different herbivores under both laboratory and field conditions. Our study revealed that both herbivore identity and parasitism affect plant transcriptional and metabolic responses to herbivory. We also found that hyperparasitoids are able to respond to HIPVs released by wild B. oleracea under both laboratory and field conditions. In addition, we observed stronger attraction of hyperparasitoids to HIPVs when plants were infested with parasitized caterpillars. However, hyperparasitoids were equally attracted to plants infested by either herbivore species. Our results indicate that parasitism plays a major role in HIPV-mediated plant-hyperparasitoid interactions. Furthermore, these findings also indicate that plant trait-mediated indirect interaction networks play important roles in community-wide species interactions.
Biofumigation using a wild Brassica oleracea accession with high glucosinolate content affects beneficial soil
Zuluaga, D.L. ; Ommen Kloeke van, A.E.E. ; Verkerk, R. ; Röling, W.F.M. ; Ellers, J. ; Roelofs, D. ; Aarts, M.G.M. - \ 2015
Plant and Soil 394 (2015). - ISSN 0032-079X - p. 155 - 163.
chemical diversity - gene-expression - indian mustard - natural toxin - life-history - isothiocyanates - collembola - release - defense - tissues
Aims This study explores the biofumigation effects of glucosinolate (GSL) containing Brassica oleracea plant material on beneficial, non-target soil organisms, and aims to relate those effects to differences in GSL profiles. Methods Leaf material of purple sprouting broccoli ‘Santee’, Savoy cabbage ‘Wintessa’, and the wild B. oleracea accession Winspit was analysed for GSL production and used for biofumigation experiments on the beneficial soil invertebrates, Folsomia candida (springtail) and Eisenia andrei (earthworm) and the soil bacterial community. Results When mixed into soil, the Winspit plant material exerted the highest toxic effects on beneficial soil invertebrates by reducing survival and reproduction. Total GSL levels varied substantially between genotypes, in particular the aliphatic GSL (AGSL) sinigrin and gluconapin being highly abundant or exclusively present in Winspit. Differences between the genotypes regarding biofumigation effects on the soil microbial community were only observed on a temporal basis with the largest difference in bacterial community structure after 1 week. Conclusions The high total GSL content in biofumigated soil could explain the toxicity of Winspit for soil invertebrates. These effects are likely to be the results of high AGSL levels in Winspit. The use of wild B. oleracea crops, such asWinspit, for biofumigation practices would need a proper assessment of the overall impact on soil biota before being applied on a wide scale
Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response
Fros, J.J. ; Major, L.D. ; Scholte, F.E. ; Gardner, J. ; Hemert, M.J. van; Suhrbier, A. ; Pijlman, G.P. - \ 2015
Journal of General Virology 96 (2015)3. - ISSN 0022-1317 - p. 580 - 589.
endoplasmic-reticulum stress - semliki-forest-virus - messenger-rna - mammalian-cells - er stress - translational shutoff - transcription factor - gene-expression - insect cells - infection
The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1a become activated. PERK phosphorylates eIF2a leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1a splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2a and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
The olfactory receptor OR51E1 is present along the gastrointestinal tract of pigs and is modulated by intestinal microbiota
Priori, D. ; Clavenzani, P. ; Jansman, A.J.M. ; Lalles, J.P. ; Trivisil, P. ; Bosi, P. - \ 2015
PLoS One 10 (2015)6. - ISSN 1932-6203 - 17 p.
enterotoxigenic escherichia-coli - butyrate-producing bacteria - fatty-acid receptor - net absorption - weaned pigs - odorant receptor - taste receptors - gene-expression - gut microbiota - serotonin
The relevance of the butyrate-sensing olfactory receptor OR51E1 for gastrointestinal (GIT) functioning has not been considered so far. We investigated in young pigs the distribution of OR51E1 along the GIT, its relation with some endocrine markers, its variation with age and after interventions affecting the gut environment and intestinal microbiota. Immuno-reactive cells for OR51E1 and chromogranin A (CgA) were counted in cardial (CA), fundic (FU), pyloric (PL) duodenal (DU), jejunal (JE), ileal (IL), cecal (CE), colonic (CO) and rectal (RE) mucosae. OR51E1 co-localization with serotonin (5HT) and peptide YY (PYY) were evaluated in PL and CO respectively. FU and PL tissues were also sampled from 84 piglets reared from sows receiving either or not oral antibiotics (amoxicillin) around parturition, and sacrificed at days 14, 21, 28 (weaning) and 42 of age. JE samples were also obtained from 12 caesarean-derived piglets that were orally associated with simple (SA) or complex (CA) microbiota in the postnatal phase, and of which on days 26–37 of age jejunal loops were perfused for 8 h with enterotoxigenic Escherichia coli F4 (ETEC), Lactobacillus amylovorus or saline (CTRL). Tissue densities of OR51E1+ cells were in decreasing order: PL=DU>FU=CA>JE=IL=CE=CO=RE. OR51E1+ cells showed an enteroendocrine nature containing gastrointestinal hormones such as PYY or 5HT. OR51E1 gene expression in PL and FU increased during and after the suckling period (p
Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields
Golbach, L.A. ; Philippi, J.G.M. ; Cuppen, J.J.M. ; Savelkoul, H.F.J. ; Verburg-van Kemenade, B.M.L. - \ 2015
Bioelectromagnetics 36 (2015)6. - ISSN 0197-8462 - p. 430 - 443.
hz magnetic-fields - cytosolic-free calcium - gene-expression - intracellular calcium - hl-60 cells - housekeeping genes - childhood leukemia - protein expression - human-lymphocytes - human monocytes
We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30¿min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50¿Hz sine wave) at three magnetic flux densities (5¿µT, 300¿µT, and 500¿µT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50¿Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.
A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem
Kinoshita, A. ; Hove, C.A. ten; Tabata, R. ; Yamada, M. ; Shimizu, N. ; Ishida, T. ; Yamaguchi, K. ; Shigenobu, S. ; Takebayashi, Y. ; Luchies, J. ; Kobayashi, M. ; Kurata, T. ; Wada, T. ; Seo, M. ; Hasebe, M. ; Blilou, I. ; Fukuda, H. ; Scheres, B. ; Heidstra, R. ; Kamiya, Y. ; Sawa, S. - \ 2015
Development 142 (2015). - ISSN 0950-1991 - p. 444 - 453.
receptor-like kinase - arabidopsis shoot meristem - of-function phenotypes - cle peptides - gene-expression - repeat protein - differentiation - thaliana - organization - growth
The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.
Variation in plant-mediated interactions between rhizobacteria and caterpillars: potential role of soil composition
Pangesti, N.P.D. ; Pineda Gomez, A.M. ; Dicke, M. ; Loon, J.J.A. van - \ 2015
Plant Biology 17 (2015)2. - ISSN 1435-8603 - p. 474 - 483.
induced systemic resistance - arbuscular mycorrhizal symbiosis - arabidopsis-thaliana - insect herbivores - gene-expression - jasmonic acid - pseudomonas-fluorescens - microbes - bacteria - immunity
Selected strains of non-pathogenic rhizobacteria can trigger induced systemic resistance (ISR) in plants against aboveground insect herbivores. However, the underlying mechanisms of plant-mediated interactions between rhizobacteria and herbivorous insects are still poorly understood. Using Arabidopsis thaliana Col-0-Pseudomonas fluorescens WCS417r as a model system, we investigated the performance and the molecular mechanisms underlying plant-mediated effects of rhizobacteria on the generalist caterpillar Mamestra brassicae and the specialist Pieris brassicae. Rhizobacteria colonisation of Arabidopsis roots resulted in decreased larval weight of M. brassicae, whereas no effect was observed on larval weight of P. brassicae. Using a jasmonic acid (JA)-impaired mutant (dde2-2), we confirmed the importance of JA in rhizobacteria-mediated ISR against M. brassicae. Interestingly, in some experiments we also observed rhizobacteria-induced systemic susceptibility to M. brassicae. The role of soil composition in the variable outcomes of microbe-plant-insect interactions was then assessed by comparing M. brassicae performance and gene transcription in plants grown in potting soil or a mixture of potting soil and sand in a 1:1 ratio. In a mixture of potting soil and sand, rhizobacteria treatment had a consistent negative effect on M. brassicae, whereas the effect was more variable in potting soil. Interestingly, at 24 h post-infestation (hpi) rhizobacteria treatment primed plants grown in a mixture of potting soil and sand for stronger expression of the JA- and ethylene-regulated genes PDF1.2 and HEL, respectively. Our study shows that soil composition can modulate rhizobacteria-plant-insect interactions, and is a factor that should be considered when studying these belowground-aboveground interactions.
The effect of quercetin and kaempferol aglycones and glucuronides on peroxisome proliferator-activated receptor-gamma (PPAR-¿)
Beekmann, K. ; Rubió, L. ; Haan, L.H.J. de; Actis Goretta, L. ; Burg, B. van der; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2015
Food & Function 6 (2015)4. - ISSN 2042-6496 - p. 1098 - 1107.
mitotic clonal expansion - in-vitro - adipocyte differentiation - dietary flavonoids - gene-expression - 3t3-l1 cells - kappa-b - mediated inflammation - cholesterol efflux - molecular target
The consumption of dietary flavonoids has been associated with a variety of health benefits, including effects mediated by the activation of peroxisome proliferator-activated receptor-gamma (PPAR-¿). Flavonoids are extensively metabolized during and after uptake and there is little known on the biological effects of these conjugated metabolites of flavonoids that are found in plasma. To investigate the effect of glucuronidation on the ability of flavonoids to activate PPAR-¿ we studied and compared the activity of quercetin, kaempferol and their relevant plasma conjugates quercetin-3-O-glucuronide (Q3G) and kaempferol-3-O-glucuronide (K3G) on different PPAR-¿ related endpoints. The flavonoid aglycones increased PPAR-¿ mediated gene expression in a stably transfected reporter gene cell line and glucuronidation diminished their effect. To study the intrinsic activity of the test compounds to activate PPAR-¿ we used a novel microarray technique to study ligand induced ligand binding domain (LBD) – nuclear receptor coregulator interactions. In this cell-free system we demonstrate that, unlike the known PPAR-¿ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the receptor. The increases in reporter gene expression in the reporter cells were accompanied by increased PPAR-¿ receptor-mRNA expression and quercetin synergistically increased the effect of rosiglitazone in the reporter gene assay. It is concluded that flavonoids affect PPAR-¿ mediated gene transcription by a mode of action different from agonist binding. Increases in PPAR-¿ receptor mRNA expression and synergistic effects with endogenous PPAR-¿ agonists may play a role in this alternative mode of action. Glucuronidation reduced the activity of the flavonoid aglycones
Fat, fibre and cancer risk in African Americans and rural Africans
O'Keefe, S.J. ; Li, J.V. ; Lahti, L.M. ; Ou, J. ; Carbonero, F. ; Mohammed, K. ; Posma, J.M. ; Kinross, J. ; Wahl, E. ; Ruder, E. ; Vipperla, K. ; Naidoo, V. ; Mtshali, L. ; Tims, S. ; Puylaert, P.G.B. ; DeLany, J. ; Krasinskas, A. ; Benefiel, A.C. ; Kaseb, H.O. ; Newton, K. ; Nicholson, J.K. ; Vos, W.M. de; Gaskins, H.R. ; Zoetendal, E.G. - \ 2015
Nature Communications 6 (2015). - ISSN 2041-1723
butyrate-producing bacteria - resistant starch - colon-cancer - cell-proliferation - phylogenetic microarray - microbial metabolites - intestinal-mucosa - epithelial-cells - gene-expression - human feces
Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (
Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line
Katika, M.R. ; Hendriksen, P.J.M. ; Loveren, H. van; Peijnenburg, A.A.C.M. - \ 2015
Journal of Immunotoxicology 12 (2015)3. - ISSN 1547-691X - p. 206 - 216.
activated protein-kinases - endoplasmic-reticulum stress - kappa-b activation - gene-expression - trichothecene deoxynivalenol - vomitoxin deoxynivalenol - transcription factors - cytokine production - induced apoptosis - wheat products
Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3¿h to 0.5¿µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-¿B (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit¿¿¿ribotoxic stress¿¿¿ER stress¿¿¿calcium release from the ER into cytoplasm¿¿¿T-cell activation and oxidative stress¿¿¿apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels.
Chemical characterization and biological activity of Chaga (Inonotus obliquus) a medicinal "mushroom"
Glamoclija, J. ; Ciric, A. ; Nikolic, M. ; Fernandes, A. ; Barros, L. ; Calhelha, R.C. ; Ferreira, I.C.F.R. ; Sokovic, M. ; Griensven, L.J.L.D. van - \ 2015
Journal of Ethnopharmacology 162 (2015). - ISSN 0378-8741 - p. 323 - 332.
pseudomonas-aeruginosa - swarming motility - in-vitro - biofilm formation - edible mushrooms - social evolution - aqueous extract - gene-expression - hot-water - antioxidant
ETHNOPHARMACOLOGICAL RELEVANCE: In Russian traditional medicine, an extract from the mushroom Inonotus obliquus (Fr.) Pil´at is used as an anti-tumor medicine and diuretic. It has been reported that Inonotus obliquus has therapeutic effects, such as anti-inflammatory, immuno-modulatory and hepatoprotective effects. This study was designed to investigate the chemical composition and biological properties of aqueous and ethanolic extracts of Inonotus obliquus from Finland, Russia, and Thailand. Their antioxidative, antimicrobial, and antiquorum properties were tested as well as the cytotoxicity on various tumor cell lines. MATERIALS AND METHODS: The tested extract was subjected to conventional chemical study to identified organic acids and phenolic compounds. Antioxidative activity was measured by several different assays. Antimicrobial potential of extracts was tested by microdilution method, and antiquorum sensing activity and antibiofilm formation of Inonotus obliquus extracts was tested on Pseudomonas aeruginosa. Cytotoxicity of the extracts was tested on tumor cells (MCF-7, NCI-H460, HeLa and HepG2) and non-tumor liver cells primary cultures. RESULTS: Oxalic acid was found as the main organic acid, with the highest amount in the aqueous extract from Russia. Gallic, protocatechuic and p-hydroxybenzoic acids were detected in all samples. Inonotus obliquus extracts showed high antioxidant and antimicrobial activity. Extracts were tested at subMIC for anti-quorum sensing (AQS) activity in Pseudomonas aeruginosa and all extracts showed definite AQS activity. The assays were done using twitching and swarming of bacterial cultures, and the amount of produced pyocyanin as QS parameters. All the extracts demonstrated cytotoxic effect on four tumor cell lines and not on primary porcine liver cells PLP2. CONCLUSIONS: As the Inonotus obliquus presence in Chaga conks is limited, further purification is necessary to draw quantitative conclusions. The presence of AQS activity in medicinal mushrooms suggests a broader anti-infectious disease protection than only immunomodulatory effects.
Structural bisphenol analoques differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor
Roelofs, M.J.E. ; Berg, M. van den; Bovee, T.F.H. ; Piersma, A.H. ; Duursen, M.B.M. van - \ 2015
Toxicology 329 (2015). - ISSN 0300-483X - p. 10 - 20.
endocrine-disrupting chemicals - tetrabromobisphenol-a tbbpa - brominated flame retardants - one-generation reproduction - in-vitro - fetal testis - exogenous progesterone - gene-expression - risk-assessment - united-states
Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67 µM, 60 µM, and 22 nM for GR, and 39 µM, 20 µM, and 982 nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestagens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular ¿4 steroidogenic pathway. Therefore, adding an in vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable.
Bright fluorescent Streptococcus pneumoniae for live cell imaging of host-pathogen interactions
Kjos, M. ; Aprianto, R. ; Fernandes, V.E. ; Andrew, P.W. ; Strijp, J.A.G. van; Nijland, R. ; Veening, J.W. - \ 2015
Journal of Bacteriology 197 (2015)5. - ISSN 0021-9193 - p. 807 - 818.
epithelial-cells - gene-expression - pneumococcal virulence - bacillus-subtilis - in-vivo - protein - capsule - colonization - disease - invasion
Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people, but at the same time one of the major causes of infectious diseases such as pneumonia, meningitis and sepsis. The shift from commensal to pathogen and its interaction with host cells is poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent and the GFP-reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells compared to encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live cell imaging of pneumococcal infection and help understand the mechanisms of pneumococcal pathogenesis.
Production of inflammatory mediators and extracellular traps by carp macrophages and neutrophils in response to lipopolysaccharide and/or interferon-¿2
Pijanowski, L. ; Scheer, M.H. ; Verburg-van Kemenade, B.M.L. ; Chadzinska, M.K. - \ 2015
Fish and Shellfish Immunology 42 (2015)2. - ISSN 1050-4648 - p. 473 - 482.
toll-like receptors - cyprinus-carpio - signaling pathways - expression analysis - escherichia-coli - immune-response - lps recognition - gene-expression - fish - l.
Neutrophilic granulocytes and macrophages are crucial for the innate immune response against infections. They migrate into the focus of inflammation, where they efficiently bind, engulf and kill bacteria by proteolytic enzymes, antimicrobial peptides, reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, activated neutrophils and macrophages can form extracellular traps (ETs). Fish neutrophils and macrophages are morphologically, histochemically, and functionally similar to their mammalian counterparts, but their significance for regulation of inflammatory responses and pathogen killing needs further elucidation. We compared the activity of head kidney monocytes/macrophages and neutrophilic granulocytes of common carp and established that upon lipopolysaccharide stimulation, not only neutrophils, but also carp monocytes/macrophages release extracellular DNA and are capable to form macrophage extracellular traps (METs). To clarify whether many specific LPS functions reported for piscine phagocytes might be due to impurities in the commonly used LPS preparations we studied expression of inflammatory mediators, release of DNA, ROS and RNS in cells stimulated with LPS or its highly purified form (pLPS). Also IFN-¿2 stimulation and its synergism with LPS/pLPS in stimulating expression of pro-inflammatory mediators was studied. Results substantiate that a classical stimulation of TLR4 by LPS may indeed be absent in carp as most of the classically reported LPS effects are abolished or diminished when pLPS is used. Interestingly, we also observed a potent IL-10 expression in neutrophilic granulocytes upon LPS stimulation, which, apart from their pro-inflammatory function, clearly indicates a role in restrictive control of the inflammatory reaction.
Evaluation of the Discriminative Potential of a Novel Biomarker for Estradiol Treatments in Bovine Animals
Regal, P. ; Blokland, M.H. ; Fente, C.A. ; Sterk, S.S. ; Cepeda, A. ; Ginkel, L.A. van - \ 2015
Journal of Agricultural and Food Chemistry 63 (2015)1. - ISSN 0021-8561 - p. 370 - 378.
tandem mass-spectrometry - timed artificial-insemination - beef-cattle - protein biomarkers - gene-expression - hormone abuse - plasma - 17-beta-estradiol - estrogen - detect
The endogenous occurrence of natural hormones obstructs the application of classical targeted methods as confirmatory options. In the case of estradiol, the ultimate confirmation of its exogenous administration relies on gas chromatography coupled to combustion/isotope ratio mass spectrometry (GC-C/IRMS). A serum dipeptide composed of pyroglutamic acid and phenylalanine was identified as a potential biomarker of estradiol treatments in adult cows. To evaluate its potential to pinpoint suspicious samples, samples from prepubertal females under different estrogenic treatments have been analyzed. The results confirmed the up-regulation of the dipeptide in adult bovines. The 2-week-old females exhibited short-lasting responses only in a few animals. The 6-month-old female showed a delayed but clear increase on the biomarker level. The composition of the anabolic preparations, the dose, and/or the administration route are possible additional reasons for the reduced response in young animals. A comparison to previous results reported by various researchers is included.
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.
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