Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Early life microbial colonization of the gut and intestinal development differ between genetically divergent broiler lines
Schokker, D. ; Veninga, G. ; Vastenhouw, S.A. ; Bossers, A. ; Bree, F.M. de; Kaal-Lansbergen, L.M.T.E. ; Rebel, J.M.J. ; Smits, M.A. - \ 2015
BMC Genomics 16 (2015). - ISSN 1471-2164
salmonella-enteritidis colonization - innate immune responsiveness - pro-inflammatory cytokine - barrier function - gastrointestinal-tract - epithelial-cells - host genotype - double-blind - chickens - resistance
Host genetic makeup plays a role in early gut microbial colonization and immune programming. Interactions between gut microbiota and host cells of the mucosal layer are of paramount importance for a proper development of host defence mechanisms. For different livestock species, it has already been shown that particular genotypes have increased susceptibilities towards disease causing pathogens. The objective of this study was to investigate the impact of genotypic variation on both early microbial colonization of the gut and functional development of intestinal tissue. From two genetically diverse chicken lines intestinal content samples were taken for microbiota analyses and intestinal tissue samples were extracted for gene expression analyses, both at three subsequent time-points (days 0, 4, and 16).
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 (
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.
Functional characterization of probiotic surface layer protein-carrying Lactobacillus amylovorus strains
Hynönen, U. ; Kant, R. ; Lähteinen, T. ; Pietilä, T.E. ; Beganovic, J. ; Smidt, H. ; Uroic, K. ; Åvall-Jääskeläinen, S. ; Palva, A. - \ 2014
BMC Microbiology 14 (2014). - ISSN 1471-2180 - 16 p.
enterotoxigenic escherichia-coli - human intestinal mucus - lactic-acid bacteria - human dendritic cells - s-layer - in-vitro - acidophilus ncfm - aeromonas-salmonicida - epithelial-cells - immune-system
Background - Adhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) –layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins. Results - Three of the L. amylovorus strains studied adhered to IPEC-1 cells, while four strains inhibited the adherence of E. coli, indicating additional mechanisms other than competition for binding sites being involved in the inhibition. None of the strains bound to porcine mucus. The culture supernatants of all of the strains exerted inhibitory effects on the growth of E. coli, Salmonella, Listeria and Yersinia, and a variable, strain-dependent induction was observed of both pro- and anti-inflammatory cytokines in human DCs. L. amylovorus DSM 16698 was shown to carry two S-layer-like proteins on its surface in addition to the major S-layer protein SlpA. In contrast to expectations, none of the major S-layer proteins of the IPEC-1 -adhering strains mediated bacterial adherence. Conclusions - We demonstrated adhesive and significant pathogen inhibitory efficacies among the swine intestinal L. amylovorus strains studied, pointing to their potential use as probiotic feed supplements, but no independent role could be demonstrated for the major S-layer proteins in adherence to epithelial cells. The results indicate that many intestinal bacteria may coexist with and confer benefits to the host by mechanisms not attributable to adhesion to epithelial cells or mucus.
Early Changes in Microbial Colonization Selectively Modulate Intestinal Enzymes, but Not Inducible Heat Shock Proteins in Young Adult Swine
Arnal, M.E. ; Zhang, J. ; Messori, S. ; Bosi, P. ; Smidt, H. ; Lallès, J.P. - \ 2014
PLoS One 9 (2014)5. - ISSN 1932-6203 - 14 p.
alkaline-phosphatase - epithelial-cells - gut microbiota - gastrointestinal-tract - gene-expression - messenger-rna - piglets - growth - diet - rat
Metabolic diseases and obesity are developing worldwide in a context of plethoric intake of high energy diets. The intestine may play a pivotal role due to diet-induced alterations in microbiota composition and increased permeability to bacterial lipopolysaccharide inducing metabolic inflammation. Early programming of metabolic disorders appearing in later life is also suspected, but data on the intestine are lacking. Therefore, we hypothesized that early disturbances in microbial colonization have short- and long-lasting consequences on selected intestinal components including key digestive enzymes and protective inducible heat shock proteins (HSP). The hypothesis was tested in swine offspring born to control mothers (n = 12) or mothers treated with the antibiotic amoxicillin around parturition (n = 11), and slaughtered serially at 14, 28 and 42 days of age to assess short-term effects. To evaluate long-term consequences, young adult offspring from the same litters were offered a normal or a fat-enriched diet for 4 weeks between 140 and 169 days of age and were then slaughtered. Amoxicillin treatment transiently modified both mother and offspring microbiota. This was associated with early but transient reduction in ileal alkaline phosphatase, HSP70 (but not HSP27) and crypt depth, suggesting a milder or delayed intestinal response to bacteria in offspring born to antibiotic-treated mothers. More importantly, we disclosed long-term consequences of this treatment on jejunal alkaline phosphatase (reduced) and jejunal and ileal dipeptidylpeptidase IV (increased and decreased, respectively) of offspring born to antibiotic-treated dams. Significant interactions between early antibiotic treatment and later diet were observed for jejunal alkaline phosphatase and sucrase. By contrast, inducible HSPs were not affected. In conclusion, our data suggest that early changes in bacterial colonization not only modulate intestinal architecture and function transiently, but also exert site- and sometimes diet-specific long-term effects on key components of intestinal homeostasis.
Sexually dimorphic characteristics of the small intestine and colon of prepubescent C57BL/6 mice
Steegenga, W.T. ; Mischke, M. ; Lute, C. ; Boekschoten, M.V. ; Pruis, M.G.M. ; Lendvai, A. ; Verkade, H.J. ; Boekhorst, J. ; Timmerman, H.M. ; Plösch, T. ; Müller, M.R. - \ 2014
Biology of Sex Differences 5 (2014). - ISSN 2042-6410 - 17 p.
liver gene-expression - inflammatory-bowel-disease - inactive x-chromosome - genome-wide analysis - sex-differences - mouse-liver - gut microbiota - pancreatic-secretion - microarray analysis - epithelial-cells
Background There is increasing appreciation for sexually dimorphic effects, but the molecular mechanisms underlying these effects are only partially understood. In the present study, we explored transcriptomics and epigenetic differences in the small intestine and colon of prepubescent male and female mice. In addition, the microbiota composition of the colonic luminal content has been examined. Methods At postnatal day 14, male and female C57BL/6 mice were sacrificed and the small intestine, colon and content of luminal colon were isolated. Gene expression of both segments of the intestine was analysed by microarray analysis. DNA methylation of the promoter regions of selected sexually dimorphic genes was examined by pyrosequencing. Composition of the microbiota was explored by deep sequencing. Results Sexually dimorphic genes were observed in both segments of the intestine of 2-week-old mouse pups, with a stronger effect in the small intestine. Amongst the total of 349 genes displaying a sexually dimorphic effect in the small intestine and/or colon, several candidates exhibited a previously established function in the intestine (i.e. Nts, Nucb2, Alox5ap and Retnl¿). In addition, differential expression of genes linked to intestinal bowel disease (i.e. Ccr3, Ccl11 and Tnfr) and colorectal cancer development (i.e. Wt1 and Mmp25) was observed between males and females. Amongst the genes displaying significant sexually dimorphic expression, nine genes were histone-modifying enzymes, suggesting that epigenetic mechanisms might be a potential underlying regulatory mechanism. However, our results reveal no significant changes in DNA methylation of analysed CpGs within the selected differentially expressed genes. With respect to the bacterial community composition in the colon, a dominant effect of litter origin was found but no significant sex effect was detected. However, a sex effect on the dominance of specific taxa was observed. Conclusions This study reveals molecular dissimilarities between males and females in the small intestine and colon of prepubescent mice, which might underlie differences in physiological functioning and in disease predisposition in the two sexes.
The Salivary Scavenger and Agglutinin in Early Life: Diverse Roles in Amniotic Fluid and in the Infant Intestine
Reichhardt, M.P. ; Jarva, H. ; Been, M. de; Rodriguez, J.M. ; Quintana, E.J. ; Loimaranta, V. ; Vos, W.M. de; Meri, S. - \ 2014
The Journal of Immunology 193 (2014)10. - ISSN 0022-1767 - p. 5240 - 5248.
surfactant protein-d - secretory immunoglobulin-a - malignant brain-tumors - terminal differentiation - bacterial recognition - streptococcus-mutans - glycoprotein gp-340 - epithelial-cells - innate immunity - receptor
The salivary scavenger and agglutinin (SALSA), also known as gp340 and dmbt1, is an antimicrobial and inflammation-regulating molecule located at the mucosal surfaces. The present study revealed that SALSA was present in the amniotic fluid (AF) and exceptionally enriched in both meconium and feces of infants. Based on immunological and mass spectrometric analysis, SALSA was estimated to constitute up to 4–10% of the total protein amount in meconium, making it one of the most abundant proteins. SALSA proteins in the AF and intestinal samples were polymorphic and exhibited varying polypeptide compositions. In particular, a different abundance of peptides corresponding to functionally important structures was found in the AF and intestinal SALSA. The AF form of SALSA had a more intact structure and contained peptides from the zona pellucida domain, which is involved in cell differentiation and oligomerization. In contrast, the intestinal SALSA was more enriched with the scavenger receptor cysteine-rich domains. The AF, but not the meconium SALSA, bound to Streptococcus pyogenes, S. agalactiae, S. gordonii, and Escherichia coli. Furthermore, differential binding was observed also to known endogenous ligands C1q, mannose-binding lectin, and secretory IgA. Our results have thus identified mucosal body compartments, where SALSA is particularly abundant, and suggest that SALSA exhibits varying functions in the different mucosal locations. The high levels of SALSA in AF and the infant intestine suggest a robust and important function for SALSA during the fetal development and in the mucosal innate immune defense of infants.
GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum
Lee, I.C. ; Swam, I.I. van; Tomita, S. ; Morsomme, P. ; Rolain, T. ; Hols, P. ; Kleerebezem, M. ; Bron, P.A. - \ 2014
Journal of Bacteriology 196 (2014)9. - ISSN 0021-9193 - p. 1671 - 1682.
complete genome sequence - lactic-acid bacteria - escherichia-coli - campylobacter-jejuni - acidophilus ncfm - epithelial-cells - surface protein - rhamnosus gg - glycoproteins - binding
Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
A lipid zipper triggers bacterial invasion
Eierhoff, T. ; Bastian, B. ; Thuenauer, R. ; Madl, J. ; Audfray, A. ; Aigal, S. ; Juillot, S. ; Rydell, G.E. ; Müller, S. ; Bentzmann, S. de; Imberty, A. ; Fleck, C. ; Römer, W. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)35. - ISSN 0027-8424 - p. 12895 - 12900.
pseudomonas-aeruginosa lectin - human endothelial-cells - epithelial-cells - host-cells - membrane invaginations - e-cadherin - pa-i - rafts - entry - receptor
Glycosphingolipids are important structural constituents of cellular membranes. They are involved in the formation of nanodomains (“lipid rafts”), which serve as important signaling platforms. Invasive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium—a key process in bacterial uptake. However, it is unknown whether glycosphingolipids directly take part in the membrane invagination process. Here, we demonstrate that a “lipid zipper,” which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3, triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aeruginosa. In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was sufficient to achieve complete membrane engulfment of the bacterium. In addition, theoretical modeling elucidated that the adhesion energy of the LecA–Gb3 interaction is adequate to drive the engulfment process. In cellulo experiments demonstrated that inhibition of the LecA/Gb3 lipid zipper by either lecA knockout, Gb3 depletion, or application of soluble sugars that interfere with LecA binding to Gb3 significantly lowered P. aeruginosa uptake by host cells. Of note, membrane engulfment of P. aeruginosa occurred independently of actin polymerization, thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry. Our study sheds new light on the impact of glycosphingolipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes.
Bluetongue virus nonstructural protein NS3/NS3a is not essential for virus replication
Gennip, H.G.P. van; Water, S.G.P. van de; Rijn, P.A. van - \ 2014
PLoS One 9 (2014)1. - ISSN 1932-6203 - 8 p.
cell-lines - epithelial-cells - infected-cells - release - trafficking - particles - systems - ns3
Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUGMet¿GCCAla mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.
Characterization of the sialic acid binding activity of Influenza A viruses using soluble variants of the H7 and H9 hemagglutinins
Sauer, A.K. ; Liang, C.H. ; Stech, J. ; Peeters, B.P.H. ; Quéré, P. ; Schwegmann-Wessels, C. ; Wu, C.Y. ; Herrler, G. - \ 2014
PLoS One 9 (2014)2. - ISSN 1932-6203
infectious-bronchitis-virus - receptor specificity - epithelial-cells - cleavage site - determinants - poultry - chicken - humans - line
Binding of influenza viruses to target cells is mediated by the viral surface protein hemagglutinin. To determine the presence of binding sites for influenza A viruses on cells and tissues, soluble hemagglutinins of the H7 and H9 subtype were generated by connecting the hemagglutinin ectodomain to the Fc portion of human immunoglobulin G (H7Fc and H9Fc). Both chimeric proteins bound to different cells and tissues in a sialic acid-dependent manner. Pronounced differences were observed between H7Fc and H9Fc, in the binding both to different mammalian and avian cultured cells and to cryosections of the respiratory epithelium of different virus host species (turkey, chicken and pig). Binding of the soluble hemagglutinins was similar to the binding of virus particles, but showed differences in the binding pattern when compared to two sialic acid-specific plant lectins. These findings were substantiated by a comparative glycan array analysis revealing a very narrow recognition of sialoglycoconjugates by the plant lectins that does not reflect the glycan structures preferentially recognized by H7Fc and H9Fc. Thus, soluble hemagglutinins may serve as sialic acid-specific lectins and are a more reliable indicator of the presence of binding sites for influenza virus HA than the commonly used plant lectins.
Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis
Ferrando, M.L. ; Baarlen, P. van; Orrù, G. ; Piga, R. ; Bongers, R.S. ; Wels, M. ; Greeff, A. de; Smith, H. ; Wells, J.M. - \ 2014
PLoS One 9 (2014)3. - ISSN 1932-6203 - 16 p.
group-a streptococcus - fibronectin-binding protein - bacillus-subtilis - glycogen-metabolism - escherichia-coli - epithelial-cells - mouse intestine - molecular characterization - hyaluronan concentration - catabolite repression
Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary a-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the a-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition
In vitro nanoparticle toxicity to rat alveolar cells and coelomocytes from the earthworm Lumbricus rubelles
Ploeg, M.J.C. van der; Berg, J.H.J. van den; Bhattacharjee, S. ; Haan, L.H.J. de; Ershov, D.S. ; Fokkink, R.G. ; Zuilhof, H. ; Rietjens, I.M.C.M. ; Brink, N.W. van den - \ 2014
Nanotoxicology 8 (2014)1. - ISSN 1743-5390 - p. 28 - 37.
neutral red retention - eisenia-foetida - celomic fluid - gold nanoparticles - epithelial-cells - terrestris - exposure - c-60 - cytotoxicity - assay
Sensitivity of immune cells (coelomocytes) of Lumbricus rubellus earthworms was investigated for exposure to selected nanoparticles, in order to obtain further insight in mechanisms of effects observed after in vivo C60 exposure. In the in vivo study, tissue damage appeared to occur without accompanying increased immune responses. Coelomocytes exposed in vitro to C60 showed no decrease of their cellular viability, but demonstrated a decrease in gene expression of the cytokine-like protein CCF-1, indicating immunosuppression. Experiments with NR8383 rat macrophage cells and tri-block copolymer nanoparticles were used to compare sensitivity and to demonstrate the usefulness of coelomocytes as a test system for nano-immunotoxicity, respectively. Overall, the results imply that sensitivity towards nanoparticles differs between cell types and nanoparticles. Moreover, this study indicates that injuries in absence of an immune response, observed after in vivo C60 exposure in our earlier work, are caused by immunosuppression rather than coelomocyte mortality.
Identification of lipid synthesis and secretion proteins in bovine milk
Lu, J. ; Hooijdonk, A.C.M. van; Boeren, S. ; Vervoort, J. ; Hettinga, K.A. - \ 2014
Journal of Dairy Research 81 (2014)1. - ISSN 0022-0299 - p. 65 - 72.
fat globule-membrane - mouse mammary-gland - proteomic analysis - acid transporters - epithelial-cells - lactation cycle - major proteins - c2 disease - cholesterol - expression
Lactation physiology is a process that is only partly understood. Proteomics techniques have shown to be useful to help advance the knowledge on lactation physiology in human and rodent species but have not been used as major tools for dairy cows, except for mastitis. In this paper, advanced non-targeted proteomics techniques (Filter aided sample preparation and NanoLC-Orbitrap-MS/MS) were applied to study the milk fat globule membrane and milk serum fraction, resulting in the identification of 246 proteins. Of these, 23 transporters and enzymes were related to lipid synthesis and secretion in mammary gland and their functions are discussed in detail. The identification of these intracellular transporters and enzymes in milk provides a possibility of using milk itself to study lipid synthesis and secretion pathways. This full-scale scan of milk proteins by using non-targeted proteomic analysis helps to reveal the important proteins involved in lipid synthesis and secretion for further examination in targeted studies.
Immunostimulatory CpG motifs in the genomes of gut bacteria and their role in human health and disease
Kant, R. ; Vos, W.M. de; Palva, A. ; Satokari, R.M. - \ 2014
Journal of Medical Microbiology 63 (2014)2. - ISSN 0022-2615 - p. 293 - 308.
toll-like receptor-9 - versus-host-disease - opportunistic pathogen - epithelial-cells - necrotizing enterocolitis - clostridium-perfringens - intestinal epithelium - multidrug-resistant - atopic disease - b-cells
Toll-like receptor (TLR) signaling plays an important role in epithelial and immune cells of the intestine. TLR9 recognizes unmethylated CpG motifs in bacterial DNA and TLR9 signaling maintains the gut epithelial homeostasis. Here, we carried out a bioinformatic analysis of the frequency of CpG motifs in the genomes of gut commensal bacteria across major bacterial phyla. The frequency of potentially immunostimulatory CpG motifs (all CpG hexamers) or PuPuCGPyPy hexamers was linearly dependent on the genomic GC content. We found that species belonging to Proteobacteria, Bacteroidetes and Actinobacteria (including bifidobacteria) carry high counts of GTCGTT, the optimal motif stimulating human TLR9. We also found that Enterococcus faecalis, Lactobacillus casei, L. plantarum and L. rhamnosus, whose strains have been marketed as probiotics, have high counts of GTCGTT motifs. As gut bacterial species differ significantly in their genomic content of CpG motifs, the overall load of CpG motifs in the intestine depends on the species assembly of microbiota and their cell numbers. The optimal CpG composition of microbiota may depend on the host's physiological status and, consequently, on the adequate level of TLR9 signaling. We speculate that microbiota with increased numbers of microbes with CpG motif-rich DNA could better support the mucosal functions in healthy individuals and improve the Th1/Th2 imbalance in allergic diseases. In autoimmune disorders, CpG motif rich DNA could, however, further increase the Th1 type immune responsiveness. Estimation of the load of microbe associated molecular patterns (MAMPs), including CpG motifs, in the gut microbiota could shed new light into host-microbe interactions across a range of diseases
Short-Chain Fatty Acids Activate AMP-Activated Protein Kinase and Ameliorate Ethanol-Induced Intestinal Barrier Dysfunction in Caco-2 Cell Monolayers
Eamin, E.E. ; Masclee, A.A. ; Dekker, J. ; Pieters, H.J. ; Jonkers, D.M. - \ 2013
The Journal of Nutrition 143 (2013)12. - ISSN 0022-3166 - p. 1872 - 1881.
alcoholic liver-disease - induced gut leakiness - paracellular permeability - signaling pathway - oxidative stress - epithelial-cells - colonic function - dietary fiber - leaky gut - butyrate
Short-chain fatty acids (SCFAs) have been shown to promote intestinal barrier function, but their protective effects against ethanol-induced intestinal injury and underlying mechanisms remain essentially unknown. The aim of the study was to analyze the influence of SCFAs on ethanol-induced barrier dysfunction and to examine the role of AMP-activated protein kinase (AMPK) as a possible mechanism using Caco-2 monolayers. The monolayers were treated apically with butyrate (2, 10, or 20 mmol/L), propionate (4, 20, or 40 mmol/L), or acetate (8, 40, or 80 mmol/L) for 1 h before ethanol (40 mmol/L) for 3 h. Barrier function was analyzed by measurement of transepithelial resistance and permeation of fluorescein isothiocyanate-labeled dextran. Distribution of the tight junction (TJ) proteins zona occludens-1, occludin, and filamentous-actin (F-actin) was examined by immunofluorescence. Metabolic stress was determined by measuring oxidative stress, mitochondrial function, and ATP using dichlorofluorescein diacetate, dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, and bioluminescence assay, respectively. AMPK was knocked down by small interfering RNA (siRNA), and its activity was assessed by a cell-based ELISA. Exposure to ethanol significantly impaired barrier function compared with controls (P <0.0001), disrupted TJ and F-actin cytoskeleton integrity, and induced metabolic stress. However, pretreatment with 2 mmol/L butyrate, 4 mmol/L propionate, and 8 mmol/L acetate significantly alleviated the ethanol-induced barrier dysfunction, TJ and F-actin disruption, and metabolic stress compared with ethanol-exposed monolayers (P <0.0001). The promoting effects on barrier function were abolished by inhibiting AMPK using either compound C or siRNA. These observations indicate that SCFAs exhibit protective effects against ethanol-induced barrier disruption via AMPK activation, suggesting a potential for SCFAs as prophylactic and/or therapeutic factors against ethanol-induced gut leakiness.
Organ specificity of beta-carotene induced lung gene-expression changes in Bcmo 1-/- mice
Helden, Y.G.J. ; Godschalk, R.W.L. ; Schooten, F.J. van; Keijer, J. - \ 2013
Molecular Nutrition & Food Research 57 (2013)2. - ISSN 1613-4125 - p. 307 - 319.
nonsteroidal antiinflammatory drugs - triple-helix repeat - retinol efficacy trial - planar cell polarity - vitamin-a - cardiovascular-disease - adipose-tissue - epidemiologic evidence - cancer prevention - epithelial-cells
Scope - Whole genome transcriptome analysis of male and female beta-carotene 15,15'-monooxygenase knockout (Bcmo1-/-) and Bcmo1+/+ (wild-type) mice with or without 14 wk of BC supplementation was done. We previously showed that only 1.8% of the genes regulated by BC in lung were also regulated in liver and inguinal white adipose tissue (iWAT), suggesting lung specific responses. Here, we explicitly questioned the lung specificity. Methods and results - We show that BC supplementation resulted in an opposite direction of gene-regulation in male compared to female Bcmo1-/- mice in lung, liver, and iWAT. This supports a systemic effect of BC on steroid hormone metabolism mediated responses. Lung, liver, and iWAT of female Bcmo1-/- mice showed an increased inflammatory response, which was counteracted by supplementation of BC. This supports a genotype dependent increased sensitivity of female mice for vitamin A deficiency. Finally, the effect of BC on Wnt signaling in male Bcmo1-/- mice was examined. Frizzled homolog 6 (Fzd6) downregulation was seen in all three tissues. Collagen triple helix containing 1 (Cthrc1) downregulation was seen in lung tissue only, suggesting specificity. Upregulation of genes involved in oxygen sensing was seen in lung and iWAT, while protocadherin upregulation was only seen in lung. Conclusion - Our results demonstrate that effects of BC are strongly sex dependent. While effects of BC on hormone metabolism mediated responses and inflammation are systemic, effects on Wnt signaling may be lung specific.
Impact of diets with a high content of greaves-meal protein or carbohydrates on faecal characteristics, volatile fatty acids and faecal calprotectin concentrations in healthy dogs
Hang, I. ; Heilmann, R.M. ; Grützner, N. ; Suchodolski, J.S. ; Steiner, J.M. ; Atroshi, F. ; Sankari, S. ; Kettunen, A. ; Vos, W.M. de; Zentek, J. ; Spillmann, T. - \ 2013
BMC Veterinary Research 9 (2013). - ISSN 1746-6148 - 8 p.
calcium-binding proteins - nutrient digestibility - intestinal inflammation - bacterial metabolites - rheumatoid-arthritis - canine calprotectin - responsive diarrhea - epithelial-cells - cystic-fibrosis - body-size
BACKGROUND: Research suggests that dietary composition influences gastrointestinal function and bacteria-derived metabolic products in the dog colon. We previously reported that dietary composition impacts upon the faecal microbiota of healthy dogs. This study aims at evaluating the dietary influences on bacteria-derived metabolic products associated with the changes in faecal microbiota that we had previously reported. We fed high-carbohydrate starch based (HCS), [crude protein: 194 g/kg, starch: 438 g/kg], high-protein greaves-meal (HPGM), [crude protein: 609 g/kg, starch: 54 g/kg] and dry commercial (DC), [crude protein: 264 g/kg, starch: 277 g/kg] diets, and studied their effects on the metabolism of the colonic microbiota and faecal calprotectin concentrations in five Beagle dogs, allocated according to the Graeco-Latin square design. Each dietary period lasted for three weeks and was crossed-over with washout periods. Food intake, body weight, and faecal consistency scores, dry matter, pH, ammonia, volatile fatty acids (VFAs), and faecal canine calprotectin concentrations were determined. RESULTS: Faecal ammonia concentrations decreased with the HCS diet. All dogs fed the HPGM diet developed diarrhoea, which led to differences in faecal consistency scores between the diets. Faecal pH was higher with the HPGM diet. Moreover, decreases in propionic and acetic acids coupled with increases in branched-chain fatty acids and valeric acid caused changes in faecal total VFAs in dogs on the HPGM diet. Faecal canine calprotectin concentration was higher with the HPGM diet and correlated positively with valeric acid concentration. CONCLUSIONS: The HPGM diet led to diarrhoea in all dogs, and there were differences in faecal VFA profiles and faecal canine calprotectin concentrations
Gut-derived short-chain fatty acids are vividly assimilated into host carbohydrates and lipids
Besten, G. den; Lange, K. ; Havinga, R. ; Dijk, T.H. van; Gerding, A. ; Eunen, K. van; Müller, M.R. ; Groen, A.K. ; Hooiveld, G.J.E.J. ; Bakker, B.M. ; Reijngoud, D.J. - \ 2013
American Journal of Physiology. Gastrointestinal and Liver Physiology 305 (2013)G900-G910. - ISSN 0193-1857
isolated rat hepatocytes - hepatic glucose-production - distal ulcerative-colitis - insulin-resistance - butyrate formation - epithelial-cells - human colon - metabolism - mice - acetate
Acetate, propionate and butyrate are the main short-chain fatty acids (SCFAs) that arise from the fermentation of fibers by the colonic microbiota. While many studies focus on the regulatory role of SCFAs, their quantitative role as a catabolic or anabolic substrate for the host has received relatively little attention. To investigate this aspect, we infused conscious mice with physiological quantities of stable isotopes [1-13C]acetate, [2-13C]propionate or [2,4-13C2]butyrate directly into the cecum, which is the natural production site in mice, and analyzed their interconversion by the microbiota as well as their metabolism by the host. Cecal interconversion - pointing to microbial cross-feeding - was high between acetate and butyrate, low between butyrate and propionate and almost absent between acetate and propionate. As much as 62% of infused propionate was used in whole-body glucose production, in line with its role as gluconeogenic substrate. Conversely, glucose synthesis from propionate accounted for 69% of total glucose production. The synthesis of palmitate and cholesterol in the liver was high from cecal acetate (2.8% and 0.7%, respectively) and butyrate (2.7% and 0.9%, respectively) as substrates, but low or absent from propionate (0.6% and 0.0%, respectively). Label incorporation due to chain elongation of stearate was approximately 8-fold higher than de novo synthesis of stearate. Microarray data suggested that SCFAs exert only a mild regulatory effect on the expression of genes involved in hepatic metabolic pathways during the 6h infusion period. Altogether, gut-derived acetate, propionate and butyrate play important roles as substrates for glucose, cholesterol and lipid metabolism.
Changes in milk proteome and metabolome associated with dry period length, energy balance and lactation stage in post parturient dairy cows
Lu, J. ; Antunes Fernandes, E.C. ; Páez Cano, A.E. ; Vinitwatanakhun, J. ; Boeren, S. ; Hooijdonk, A.C.M. van; Knegsel, A.T.M. van; Vervoort, J. ; Hettinga, K.A. - \ 2013
Journal of Proteome Research 12 (2013)7. - ISSN 1535-3893 - p. 3288 - 3296.
mouse mammary-gland - epithelial-cells - fatty-acids - inflammatory response - beta-hydroxybutyrate - ketone-bodies - liver - apoptosis - stomatin - health
The early lactation period of dairy cows, which produce high quantities of milk, is normally characterized by an insufficient energy intake to cover milk production and maintenance requirements. Mobilization of body reserves occurs to compensate this negative energy balance (NEB), and probably as a consequence there is a higher susceptibility to diseases and metabolic disorders. There are several diagnostic methods to detect NEB, usually involving ketosis related parameters. Due to the easy availability of milk this is a preferred matrix, but simple and robust predictors of NEB level are missing. To better understand the physiological mechanism of NEB, milk of cows subjected to different dry period lengths, in different energy balance status and lactation stage, were analyzed by untargeted metabolomics and proteomics techniques. Milk of cows in severe NEB showed higher concentrations of acute phase response proteins, unsaturated fatty acids, and galactose-1-phosphate. Improved energy balance (EB) resulted in higher concentration of cholesterol, cholesterol synthesis related proteins, and stomatin. The presence of stomatin and galactose-1-phosphate in milk was strongly dependent on the EB of the cows. These novel and interesting findings warrant more in-depth research to assess their applicability as robust indicators of NEB in milk and to clarify the role of stomatin and galactose-1-phophate in milk of dairy cows in NEB.
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