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
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
Carryover of cadmium from feed in growing pigs
Hoogenboom, L.A.P. ; Hattink, J. ; Polanen, A. van; Oostrom, J.J. van; Verbunt, J.T. ; Traag, W.A. ; Kan, K.A. ; Eijkeren, J.C.H. ; Boeck, G. de; Zeilmaker, M.J. - \ 2015
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 32 (2015)1. - ISSN 1944-0049 - p. 68 - 79.
cd-saturation method - sludge-amended soil - metallothionein - tissues - growth - swine - accumulation - dioxins - protein - cortex
Growing male pigs were exposed to cadmium (Cd) at levels around 1 and 10 mg kg–1 feed for up to 12 weeks, administered as CdCl2 or Cd-cysteine (CdCys). Pigs exposed to 10 mg kg–1 showed decreased growth during the last 3 weeks. Liver and kidney concentrations of Cd continuously increased over the entire 12-week exposure, exceeding the European Union limits of 1.0 mg kg–1 (kidney) and 0.5 mg kg–1 (liver) within 3 weeks at the feed level of 10 mg kg–1. A switch to clean feed after 3 weeks for 5 or 9 weeks resulted in steadily decreased levels in kidney and liver, which could be completely attributed to organ growth. At the lower feed level, the level in kidney exceeded the limit almost twofold after 12 weeks, but not after 3 weeks. Liver levels remained below the limit. Metallothionein (MT) levels in livers showed a steady decrease in both untreated and treated animals over time. In kidney such a decrease was only observed in control animals, whereas in the highest-dosed animals the MT concentrations steadily increased. The observed carryover of Cd from feed to liver and kidney was modelled by means of a simple transfer model relating levels in feed via MT levels to accumulation of Cd. Using this model, it was shown that the exposure period of growing pigs to feed containing the European Union limit of 0.5 mg kg–1 feed should be less than 12 weeks in order to prevent Cd levels in the kidneys to exceed the European Union limit.
Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin
Fereidouni, F. ; Bader, A.N. ; Colonna, A. ; Gerritsen, H.C. - \ 2014
Journal of Biophotonics 7 (2014)8. - ISSN 1864-063X - p. 589 - 596.
mouse skin - microscopy - tomography - resolution - collagen - nad(p)h - tissues
Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited auto-fluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content.
Evaluation of two commercial, rapid, ELISA kits testing or scrapie in retro-pharyngeal lymph nodes in sheep
Kittelberger, R. ; McIntuyre, L. ; Watts, S. ; MacDiarmid, S. ; Hannah, M.J. ; Jenner, J. ; Bueno, R. ; Swainsbury, R. ; Langeveld, J.P.M. ; Keulen, L.J.M. van; Zijderveld, F.G. van; Wemheuer, W.M. ; Richt, J.A. ; Sorenson, S.J. ; Pigott, C.J. ; O'Keefe, J.S. - \ 2014
New Zealand Veterinary Journal 62 (2014)6. - ISSN 0048-0169 - p. 343 - 350.
natural scrapie - prion protein - immunohistochemical detection - new-zealand - prp - accumulation - diagnosis - genotypes - tissues - brain
AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes. METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID). RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3-75.3)% for ELISA-BR and 91.6 (95% CI=88.2-94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID. CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity.
A physiologically based kinetic (PBK) model describing plasma concentrations of quercetin and its metabolites in rats
Boonpawa, R. ; Spenkelink, A. ; Rietjens, I. ; Punt, A. - \ 2014
Biochemical Pharmacology 89 (2014)2. - ISSN 0006-2952 - p. 287 - 299.
flavonoid-mediated inhibition - blood partition-coefficients - oral bioavailability - biological-activity - biliary-excretion - intestinal uptake - glucuronidation - humans - absorption - tissues
Biological activities of flavonoids in vivo are ultimately dependent on the systemic bioavailability of the aglycones as well as their metabolites. In the present study, a physiologically based kinetic (PBK) model was developed to predict plasma concentrations of the flavonoid quercetin and its metabolites and to tentatively identify the regiospecificity of the major circulating metabolites. The model was developed based on in vitro metabolic parameters and by fitting kinetic parameters to literature available in vivo data. Both exposure to quercetin aglycone and to quercetin-4'-O-glucoside, for which in vivo data were available, were simulated. The predicted plasma concentrations of different metabolites adequately matched literature reported plasma concentrations of these metabolites in rats exposed to 4'-O-glucoside. The bioavailability of aglycone was predicted to be very low ranging from 0.004%-0.1% at different oral doses of quercetin or quercetin-4'-O-glucoside. Glucuronidation was a crucial pathway that limited the bioavailability of the aglycone, with 95–99% of the dose being converted to monoglucuronides within 1.5–2.5 h at different dose levels ranging from 0.1 to 50 mg/kg bw quercetin or quercetin-4'-O-glucoside. The fast metabolic conversion to monoglucuronides allowed these metabolites to further conjugate to di- and tri-conjugates. The regiospecificity of major circulating metabolites was observed to be dose-dependent. As we still lack in vivo kinetic data for many flavonoids, the developed model has a great potential to be used as a platform to build PBK models for other flavonoids as well as to predict the kinetics of flavonoids in humans.
Macroscopic and microscopic spatially-resolved analysis of food contaminants and constituents using laser-ablation electrospray ionization mass spectrometry imaging
Nielen, M.W.F. ; Beek, T.A. van - \ 2014
Analytical and Bioanalytical Chemistry 406 (2014)27. - ISSN 1618-2642 - p. 6805 - 6815.
in-vivo - local analysis - tissues - ms
Laser-ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI) does not require very flat surfaces, high-precision sample preparation, or the addition of matrix. Because of these features, LAESI-MSI may be the method of choice for spatially-resolved food analysis. In this work, LAESI time-of-flight MSI was investigated for macroscopic and microscopic imaging of pesticides, mycotoxins, and plant metabolites on rose leaves, orange and lemon fruit, ergot bodies, cherry tomatoes, and maize kernels. Accurate mass ion-map data were acquired at sampling locations with an x–y center-to-center distance of 0.2–1.0 mm and were superimposed onto co-registered optical images. The spatially-resolved ion maps of pesticides on rose leaves suggest co-application of registered and banned pesticides. Ion maps of the fungicide imazalil reveal that this compound is only localized on the peel of citrus fruit. However, according to three-dimensional LAESI-MSI the penetration depth of imazalil into the peel has significant local variation. Ion maps of different plant alkaloids on ergot bodies from rye reveal co-localization in accordance with expectations. The feasibility of using untargeted MSI for food analysis was revealed by ion maps of plant metabolites in cherry tomatoes and maize-kernel slices. For tomatoes, traveling-wave ion mobility (TWIM) was used to discriminate between different lycoperoside glycoalkaloid isomers; for maize quadrupole time-of-flight tandem mass spectrometry (MS–MS) was successfully used to elucidate the structure of a localized unknown. It is envisaged that LAESI-MSI will contribute to future research in food science, agriforensics, and plant metabolomics.
Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie
Filali, H. ; Martin-Burriel, I. ; Harders, F. ; Varona, L. ; Hedman, C. ; Mediano, D.R. ; Monzón, M. ; Bossers, A. ; Badiola, J.J. - \ 2014
BMC Genomics 15 (2014). - ISSN 1471-2164 - 17 p.
carboxyl-terminal hydrolase - central-nervous-system - infected mouse brains - alzheimers-disease - prion protein - parkinsons-disease - identification - pathogenesis - tissues - prpsc
Background - Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. Results - In the clinical stage of the disease, we detected 105 genes that were differentially expressed (=2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. Conclusions - The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.
The PIN family of proteins in potato and their putative role in tuberisation
Roumeliotis, E. ; Kloosterman, B.A. ; Oortwijn, M.E.P. ; Visser, R.G.F. ; Bachem, C.W.B. - \ 2013
Frontiers in Plant Science 4 (2013). - ISSN 1664-462X
arabidopsis-thaliana - auxin biosynthesis - root gravitropism - tuber initiation - expression - transport - growth - identification - transformation - tissues
The PIN family of trans-membrane proteins mediates auxin efflux throughout the plant and during various phases of plant development. In Arabidopsis thaliana, the PIN family comprised of 8 members, divided into ‘short’ and ‘long’ PINs according to the length of the hydrophilic domain of the protein. Based on sequence homology using the recently published potato genome sequence (Solanum tuberosum group Phureja) we identified ten annotated potato StPIN genes. Mining the publicly available gene expression data, we constructed a catalogue tissue specificity of StPIN gene expression, focusing on the process of tuberization. A total of four StPIN genes exhibited increased expression four days after tuber induction, prior to the onset of stolon swelling. For two PIN genes, StPIN4 and StPIN2, promoter sequences were cloned and fused to the GUS reporter protein to study tissue specificity in more detail. StPIN4 promoter driven GUS staining was detected in the flower stigma, in the flower style, below the ovary and petals, in the root tips, in the vascular tissue of the stolons and in the tuber parenchyma cells. StPIN2 promoter driven GUS staining was detected in flower buds, in the vascular tissue of the swelling stolons and in the storage parenchyma of the growing tubers. Based on our results, we postulate a role for the StPINs in redistributing auxin in the swelling stolon during early events in tuber development.
Confirmation and 3D profiling of anabolic steroid esters in injection sites using imaging desorption electrospray ionisation (DESI) mass spectrometry
Rijke, E. de; Hooijerink, H. ; Sterk, S.S. ; Nielen, M.W.F. - \ 2013
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 30 (2013)6. - ISSN 1944-0049 - p. 1012 - 1019.
tissues - cocktails - brain - drugs
In this study, desorption electrospray ionization (DESI) linear ion trap tandem mass spectrometry (MSn) was applied for the confirmation and 3D profiling of anabolic steroid esters in an injection site of bovine muscle. The spatial resolution of the DESI-MSn was demonstrated by scanning hormone esters and marker ink lines drawn at various distances on a microscopic slide at set distances, using an x-scanner with manual y and z adjustment. Tissue slices of bovine muscle injected with a hormone cocktail were analysed. All anabolic steroid esters could be directly detected in the sample and confirmed based on identification points awarded for selected MS/MS transitions according to the performance criteria in Commission Decision 2002/657/EC. Moreover, the injection site could be mapped by 2D and 3D imaging MS, showing a horizontal and vertical distribution through the muscle tissue. This DESI approach offers potential for analysis of injection sites of steroid esters from illegally treated animals; moreover, direct analysis by ambient imaging DESI-MS still allows conventional extraction and analysis of the whole tissue for further confirmatory or contraanalysis afterwards.
The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria
Bosma, M. ; Minnaard, R. ; Sparks, L.M. ; Schaart, G. ; Losen, M. ; Baets, M.H. de; Duimel, H. ; Kersten, A.H. ; Bickel, P.E. ; Schrauwen, P. ; Hesselink, M.K.C. - \ 2012
Histochemistry and Cell Biology 137 (2012)2. - ISSN 0948-6143 - p. 205 - 216.
carnitine-palmitoyltransferase-i - human skeletal-muscle - substrate metabolism - lipolysis - adipocytes - peptides - promotes - tissues - family - signal
Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.
Laser-induced breakdown spectroscopy for the study of the pattern of silicon deposition in leaves of saccharum species
Tripathi, D.K. ; Kumar, R. ; Chauhan, D.K. ; Rai, A.K. ; Bicanic, D.D. - \ 2011
Instrumentation Science and Technology 39 (2011)6. - ISSN 1073-9149 - p. 510 - 521.
metal accumulation - plant materials - tissues
The spatial distribution pattern of silicon in the leaves of three species of Saccharum has been demonstrated by means of laser induced breakdown spectroscopy (LIBS). The in-situ point detection capability of LIBS was used to determine different elements in leaf samples. The concentrations of silicon and other elements in different parts of the leaves were estimated by measuring the intensities of their corresponding atomic lines. Silicon, deposited in the form of phytoliths, was also isolated by using the dry ash technique. LIBS observations showed that in all three Saccharum species, the concentration of silicon was highest in the midrib followed by that found in margin and vein areas. The concentration of silicon in S. officinarum is higher in comparison to S. spontaneum and S. bengalense. Furthermore, the concentration of silicon at the upper surface of the leaf was larger than at the lower surface. The LIBS spectra of Saccharum species also show the presence of spectral lines of Na, Mg, Ca, Fe, and K.
Isolation and characterization of strong gene regulatory sequences from apple, Malus x domestica
Schaart, J.G. ; Tinnenbroek, I.E.M. ; Krens, F.A. - \ 2011
Tree Genetics and Genomes 7 (2011)1. - ISSN 1614-2942 - p. 135 - 142.
rbcs-3a gene - expression - plants - elements - agrobacterium - arabidopsis - cisgenesis - resistance - promoters - tissues
For the strong expression of genes in plant tissue, the availability of specific gene regulatory sequences is desired. We cloned promoter and terminator sequences of an apple (Malus x domestica) ribulose biphosphate carboxylase small subunit gene (MdRbcS), which is known for its high expression and used gus reporter gene expression to test the regulatory activity of the isolated promoter and terminator sequences in transgenic tobacco. The MdRbcS promoter itself seemed to be less strong than the CaMV35S promoter when both used in combination with the nos terminator. However, the combination of the promoter and terminator of MdRbcS was able to drive gus to similar expression levels as the reference construct with CaMV35S promoter and nos terminator. This observation indicates the importance of the terminator sequence for gene expression. It is concluded that the combination of the MdRbcS promoter and terminator is a suitable regulatory sequence set for the expression of transgenes to a high level in plants and for intragenesis in apple specifically
Agonistic and antagonistic estrogens in licorice root (Glycyrrhiza glabra)
Simons, R. ; Vincken, J.P. ; Mol, L.A.M. ; The, S.A.M. ; Bovee, T.F.H. ; Luijendijk, T.J.C. ; Verbruggen, M.A. ; Gruppen, H. - \ 2011
Analytical and Bioanalytical Chemistry 401 (2011)1. - ISSN 1618-2642 - p. 305 - 313.
breast-cancer cells - antiestrogenic activities - receptor-alpha - phenolic-compounds - in-vitro - constituents - mcf-7 - flavonoids - tissues - protein
The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular, prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by liquid chromatography–mass spectrometry and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptors (ERs; ERa and ERß). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERa. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17ß-estradiol (E2). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20–60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERa or ERß subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E2, not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yeast-enhanced green fluorescent protein, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERa-selective antagonism, similar to the ERa-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E2 by approximately 80% at 6¿×¿10-6 M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERa.
Intrusive growth of sclerenchyma fibers
Snegireva, A.V. ; Ageeva, M.V. ; Amenitskii, S.I. ; Chernova, T.E. ; Ebskamp, M. ; Gorshkova, T.A. - \ 2010
Russian Journal of Plant Physiology 57 (2010)3. - ISSN 1021-4437 - p. 342 - 355.
plant-cell wall - gnetum-gnemon gnetales - microarray analysis - functional genomics - wood formation - phloem fibers - differentiation - tissues - flax - elongation
Intrusive growth is a type of cell elongation when the rate of its longitudinal growth is higher than that of surrounding cells; therefore, these cells intrude between the neighboring cells penetrating the middle lamella. The review considers the classical example of intrusive growth, e.g., elongation of sclerenchyma fibers when the cells achieve the length of several centimeters. We sum the published results of investigations of plant fiber intrusive growth and present some features of intrusive growth characterized by the authors for flax (Linum usitatissimum L.) and hemp (Cannabis sativa L.) fibers. The following characteristics of intrusive growth are considered: its rate and duration, relationship with the growth rate of surrounding cells, the type of cell elongation, peculiarities of the fiber primary cell wall structure, fibers as multinucleate cells, and also the control of intrusive growth. Genes, which expression is sharply reduced at suppression of intrusive growth, are also considered. Arguments for separation of cell elongation and secondary cell wall formation in phloem fibers and also data indicating diffuse type of cell enlargement during intrusive growth are presented.
Feasibility of a liver transcriptomics approach to assess bovine treatment with the prohormone dehydroepiandrosterone (DHEA)
Rijk, J.C.W. ; Peijnenburg, A.A.C.M. ; Hendriksen, P.J.M. ; Hende, J. van; Groot, M.J. ; Nielen, M.W.F. - \ 2010
BMC Veterinary Research 6 (2010). - ISSN 1746-6148
gene-expression-biomarkers - messenger-rna expression - anabolic agents - androgen - tissues - muscle - abuse - sport
Background Within the European Union the use of growth promoting agents in animal production is prohibited. Illegal use of natural prohormones like dehydroepiandrosterone (DHEA) is hard to prove since prohormones are strongly metabolized in vivo. In the present study, we investigated the feasibility of a novel effect-based approach for monitoring abuse of DHEA. Changes in gene expression profiles were studied in livers of bull calves treated orally (PO) or intramuscularly (IM) with 1000 mg DHEA versus two control groups, using bovine 44K DNA microarrays. In contrast to controlled genomics studies, this work involved bovines purchased at the local market on three different occasions with ages ranging from 6 to 14 months, thereby reflecting the real life inter-animal variability due to differences in age, individual physiology, season and diet. Results As determined by principal component analysis (PCA), large differences in liver gene expression profiles were observed between treated and control animals as well as between the two control groups. When comparing the gene expression profiles of PO and IM treated animals to that of all control animals, the number of significantly regulated genes (p-value 1.5) was 23 and 37 respectively. For IM and PO treated calves, gene sets were generated of genes that were significantly regulated compared to one control group and validated versus the other control group using Gene Set Enrichment Analysis (GSEA). This cross validation, showed that 6 out of the 8 gene sets were significantly enriched in DHEA treated animals when compared to an 'independent' control group. ConclusionThis study showed that identification and application of genomic biomarkers for screening of (pro)hormone abuse in livestock production is substantially hampered by biological variation. On the other hand, it is demonstrated that comparison of pre-defined gene sets versus the whole genome expression profile of an animal allows to distinguish DHEA treatment effects from variations in gene expression due to inherent biological variation. Therefore, DNA-microarray expression profiling together with statistical tools like GSEA represent a promising approach to screen for (pro)hormone abuse in livestock production. However, a better insight in the genomic variability of the control population is a prerequisite in order to define growth promoter specific gene sets that can be used as robust biomarkers in daily practice.
Gene Expression in Chicken Reveals Correlation with Structural Genomic Features and Conserved Patterns of Transcription in the Terrestrial Vertebrates
Nie, H. ; Crooijmans, R.P.M.A. ; Lammers, A. ; Schothorst, E.M. van; Keijer, J. ; Neerincx, P. ; Leunissen, J.A.M. ; Megens, H.J.W.C. ; Groenen, M.A.M. - \ 2010
PLoS ONE 5 (2010)8. - ISSN 1932-6203 - 7 p.
human housekeeping genes - salmonella infection - eimeria-maxima - lines - responses - selection - evolution - intestine - clusters - tissues
Background - The chicken is an important agricultural and avian-model species. A survey of gene expression in a range of different tissues will provide a benchmark for understanding expression levels under normal physiological conditions in birds. With expression data for birds being very scant, this benchmark is of particular interest for comparative expression analysis among various terrestrial vertebrates. Methodology/Principal Findings - We carried out a gene expression survey in eight major chicken tissues using whole genome microarrays. A global picture of gene expression is presented for the eight tissues, and tissue specific as well as common gene expression were identified. A Gene Ontology (GO) term enrichment analysis showed that tissue-specific genes are enriched with GO terms reflecting the physiological functions of the specific tissue, and housekeeping genes are enriched with GO terms related to essential biological functions. Comparisons of structural genomic features between tissue-specific genes and housekeeping genes show that housekeeping genes are more compact. Specifically, coding sequence and particularly introns are shorter than genes that display more variation in expression between tissues, and in addition intergenic space was also shorter. Meanwhile, housekeeping genes are more likely to co-localize with other abundantly or highly expressed genes on the same chromosomal regions. Furthermore, comparisons of gene expression in a panel of five common tissues between birds, mammals and amphibians showed that the expression patterns across tissues are highly similar for orthologuous genes compared to random gene pairs within each pair-wise comparison, indicating a high degree of functional conservation in gene expression among terrestrial vertebrates. Conclusions - The housekeeping genes identified in this study have shorter gene length, shorter coding sequence length, shorter introns, and shorter intergenic regions, there seems to be selection pressure on economy in genes with a wide tissue distribution, i.e. these genes are more compact. A comparative analysis showed that the expression patterns of orthologous genes are conserved in the terrestrial vertebrates during evolution
Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR
Bigarre, L. ; Baud, M. ; Cabon, J. ; Antychowicz, J. ; Bergmann, S.M. ; Engelsma, M.Y. ; Pozet, F. ; Reichert, M. ; Castric, J. - \ 2009
Journal of Virological Methods 158 (2009)1-2. - ISSN 0166-0934 - p. 51 - 57.
simplex-virus type-1 - polymerase-chain-reaction - koi herpesvirus - common carp - thymidine kinase - infected fish - dna - mortality - disease - tissues
To date, all the isolates of Cyprinid herpesvirus type-3 (CyHV3) responsible for serious outbreaks in carps Cyprinus carpio have been found to be very similar or identical on the basis of DNA sequences of a few reference genes. However, two genetic lineages (U/I and J) are distinguished by full-length genome sequencing. Two molecular markers presenting genetic variations were targeted for developing a duplex PCR assay able to distinguish CyHV3-U/I from CyHV3-J while avoiding DNA sequencing. The method was validated on a series of 42 samples of infected carps from France, The Netherlands and Poland collected from 2001 to 2008. Among these samples, both the U/I and J genotypes were identified, but also a third genotype representing a genetic intermediate between U/I and J for one of the two molecular markers. A classification of CyHV3 genotypes, based on the alleles of the two molecular markers, is proposed. The assay is easy to perform and provides a genotype information with samples moderately or highly concentrated. This tool should improve our knowledge regarding the present distribution and future diversification of this emerging virus.
Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model
Higuera-Sierra, G. ; Schop, D. ; Janssen, F. ; Dijkhuizen-Radersma, R. ; Boxtel, A.J.B. van; Blitterswijk, C.A. van - \ 2009
Tissue Engineering. Part A 15 (2009)9. - ISSN 1937-3341 - p. 2653 - 2663.
marrow stromal cells - mammalian-cells - bone-marrow - culture - expansion - glutamine - density - tissues
Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100cells/cm2 than when seeding at 1000cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17–32h, specific glucose consumption in the order of 1.25×10-1 to 3.77×10-1pmol/cell/h, specific lactate production in the order of 2.48×10-1 to 7.67×10-1pmol/cell/h, specific glutamine production in the order of 7.04×10-3 to 2.27pmol/cell/h, and specific glutamate production in the order of 4.87×10-1 to 23.4pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitro
A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products
Reece, P. ; Bremer, M.G.E.G. ; Stones, R. ; Danks, C. ; Baumgartner, S. ; Tomkies, V. ; Hemetsberger, C. ; Smits, N.G.E. ; Lubbe, W. - \ 2009
Analytical and Bioanalytical Chemistry 394 (2009)7. - ISSN 1618-2642 - p. 1845 - 1851.
A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further study