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.

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Data-Driven Modeling of Intracellular Auxin Fluxes Indicates a Dominant Role of the ER in Controlling Nuclear Auxin Uptake
Middleton, Alistair M. ; Bosco, Cristina Dal; Chlap, Phillip ; Bensch, Robert ; Harz, Hartmann ; Ren, Fugang ; Bergmann, Stefan ; Wend, Sabrina ; Weber, Wilfried ; Hayashi, Ken Ichiro ; Zurbriggen, Matias D. ; Uhl, Rainer ; Ronneberger, Olaf ; Palme, Klaus ; Fleck, Christian ; Dovzhenko, Alexander - \ 2018
Cell Reports 22 (2018)11. - ISSN 2211-1247 - p. 3044 - 3057.
auxin - auxin flux - auxin sensor - endoplasmic reticulum - fluorescent aux - mathematical modeling - microscopy - nucleus - protoplasts - single cells
In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells. Middleton et al. study how the plant phytohormone auxin enters the nucleus by using quantitative phenotyping in single plant cell systems and bespoke mathematical models that relate controlled perturbations to experimentally measurable responses. Their findings show that auxin predominantly enters the nucleus via the endoplasmic reticulum.
Controlling the self-assembly of protein polymers via heterodimer-forming modules
Domeradzka, Natalia Eliza - \ 2016
University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - massaspectrometrie - microscopie - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie

Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.

The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.

In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.

Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.

A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.

Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.

In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.

IAG ring test animal proteins 2016
Raamsdonk, L.W.D. van; Rhee, N.E. van de; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Vliege, J.J.M. ; Pinckaers, V.G.Z. - \ 2016
Wageningen : RIKILT Wageningen UR (RIKILT report 2016.008) - 31 p.
ring test - animal proteins - analytical methods - microscopy - fish feeding - animal health - polymerase chain reaction - ringtest - dierlijke eiwitten - analytische methoden - microscopie - visvoeding - diergezondheid - polymerase-kettingreactie
The annual ring test for the detection of animal proteins in animal feed of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy was organized by RIKILT - Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method. The current 2016 version of the IAG ring test for animal proteins facilitated the full scenario with the methods for microscopy and PCR as published in Regulation (EC) 51/2013 amending Annex VI of Regulation (EC) 152/2009 together with accompanying SOPs. All four samples were based on an artificial feed mimicking a formulation for ruminant feed. Two samples were labelled as fish feed (B and D), which was effectuated by adding 2% of a general fish meal. Adulteration was achieved by adding 0.1% pig MBM (B), 0.1% ruminant MBM (D) and a combination of 0.1% ruminant MBM and 0.1% fish meal (C). This combination of different spikes allowed the diverse application of the detection methods. Forty eight participants enrolled for the ring test, of which 45 submitted microscopic results. Of these, 20 participants applied the combination of microscopic and PCR analysis. Three participants submitted exclusively PCR results.
Krachtpatsers in de kas
Goud, J.C. - \ 2015
Gewasbescherming 46 (2015)4. - ISSN 0166-6495 - p. 122 - 123.
tuinbouw - glastuinbouw - biologische bestrijding - chemische bestrijding - sierteelt - chrysanthemum - microscopie - tentoonstellingen - insectenbestrijding - thrips - frankliniella occidentalis - nematoda - steinernema feltiae - horticulture - greenhouse horticulture - biological control - chemical control - ornamental horticulture - microscopy - exhibitions - insect control
Op 3 september 2015 opende het museum Micropia, samen met BASF, een nieuwe opstelling over biologische gewasbescherming. De tentoonstelling ‘Krachtpatsers in de kas’ laat het grote publiek zien hoe microscopisch kleine insectenjagers worden ingezet bij de bestrijding van trips in de sierteelt. Californische trips (Frankliniella occidentalis) is een moeilijk te bestrijden probleem in veel kasgewassen. De insecten zijn zeer klein en kruipen weg in de groeipunten van het gewas. Hierdoor ontsnappen ze vaak aan chemische middelen.
IAG ring test feed composition 2015
Raamsdonk, L.W.D. van; Rhee, N.E. van de; Pinckaers, V.G.Z. ; Vliege, J.J.M. - \ 2015
RIKILT Wageningen UR (RIKILT report 2015.017) - 25 p.
feed formulation - pig feeding - ring test - microscopy - voersamenstelling - varkensvoeding - ringtest - microscopie
A ring test was organized for the microscopic determination of botanic composition in animal feed in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the ring test was RIKILT Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the method for composition analysis of feed.
IAG ring test animal proteins 2015
Raamsdonk, L.W.D. van; Rhee, N.E. van de; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Vliege, J.J.M. ; Pinckaers, V.G.Z. - \ 2015
Wageningen : RIKILT Wageningen UR (RIKILT report 2015.016) - 31 p.
ring test - microscopy - animal proteins - cattle feeding - pig feeding - animal health - ringtest - microscopie - dierlijke eiwitten - rundveevoeding - varkensvoeding - diergezondheid
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the ring test was RIKILT - Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method. The current 2015 version of the IAG ring test for animal proteins is the first one in the IAG series of ring tests applying the full new method for microscopy as published in Regulation (EC) 51/2013 amending Annex VI of Regulation (EC) 152/2009 together with accompanying SOPs.
Permeability of gels is set by the impulse applied on the gel
Urbonaite, V. ; Jongh, H.H.J. de; Linden, E. van der; Pouvreau, L.A.M. - \ 2015
Food Hydrocolloids 50 (2015). - ISSN 0268-005X - p. 7 - 15.
globular protein gels - blood-plasma gels - ionic-strength - water - ph - microscopy - rheology - polyacrylamide - pressure - gelation
To better understand sensory perception of foods, water exudation studies on protein-based gels are of a high importance. It was aimed to study the interplay of gel coarseness and gel stiffness on water holding (WH) and water flow kinetics from the gel once force is applied onto the material. Ovalbumin heat-set gels were used as a model system, where protein volume fraction was kept constant and ionic strength was varied to obtain a range of different gel morphologies and stiffness. WH of gels was measured both as a function of time and force applied. From experimental data (i) an effective gel permeability coefficient and (ii) an effective water flux coefficient were obtained and related to gel coarseness and stiffness. Gel coarseness determined maximum amount of water removed from the gel at defined conditions, where lower (=0.1 µm) and upper (=0.4 µm) limiting scales for water removal were identified. Gel stiffness is the major determinant for water removal kinetics from the gel. The combination of gel coarseness and gel stiffness showed a cooperative effect on gel WH. The insights can be exploited in product development to predict and tune oral perception properties of (new) products.
Surface characterization and antifouling properties of nanostructured gold chips for imaging surface plasmon resonance biosensing
Joshi, S. ; Pellacani, P. ; Beek, T.A. van; Zuilhof, H. ; Nielen, M.W.F. - \ 2015
Sensors and Actuators B: Chemical 209 (2015). - ISSN 0925-4005 - p. 505 - 514.
mass-spectrometry - nonspecific adsorption - zwitterionic polymers - organic monolayers - films - spr - functionalization - immobilization - microscopy - proteins
Surface Plasmon Resonance (SPR) optical sensing is a label-free technique for real-time monitoring of biomolecular interactions. Recently, a portable imaging SPR (iSPR) prototype instrument, featuring a nanostructured gold chip, has been developed. In the present work, we investigated the crucial first steps, prior to eventual use of the nanostructured iSPR chip, i.e., its surface modification, in-depth surface characterization and the antifouling performance. Results were compared with conventional flat (i)SPR gold chips having the same surface chemistries, viz. different types of polyethylene glycol and zwitterionic polymers. Characterization of the (i)SPR chips before and after surface modification was performed using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA), X-ray photoelectron spectroscopy (XPS) and direct analysis in real time high resolution mass spectrometry (DART-HRMS). The antifouling properties were then studied using the nanostructured chip in the portable iSPR instrument and the flat gold chip in conventional SPR setup. The zwitterionic polymer surface chemistries showed the best antifouling properties. Comparison of the nanostructured iSPR chips with conventional flat (i)SPR gold chips showed that the latter perform slightly better in terms of surface modification as well as antifouling properties. The portable iSPR instrument is almost as sensitive as conventional iSPR (IBIS) and nine times less sensitive than conventional SPR (Biacore 3000). The nanostructured iSPR chip, along with the portable instrument, offers the advantage of about ten-fold reduction in instrument size, weight and costs compared to conventional (i)SPR instruments using flat gold, thus making it highly interesting for future biosensing applications.
Post-Travel Screening of Asymptomatic Long-Term Travelers to the Tropics for Intestinal Parasites Using Molecular Diagnostics
Soonawala, D. ; Lieshout, L. ; Boer, M.A.M. den; Claas, E.C.J. ; Verweij, J.J. ; Godkewitsch, A. ; Ratering, M. ; Visser, L.G. - \ 2014
American Journal of Tropical Medicine and Hygiene 90 (2014)5. - ISSN 0002-9637 - p. 835 - 839.
real-time pcr - strongyloides-stercoralis - laboratory tests - fecal samples - netherlands - microscopy - infection - diarrhea - protozoa
The incidence of asymptomatic travel-related parasitic infection is uncertain. Previous studies did not distinguish new incident infections, from past infections. Regardless of symptoms, we performed multiplex real-time polymerase chain reaction on pre- and post-travel stool samples of Dutch long-term travelers to the (sub)tropics. Serological screening for Schistosoma spp. was only performed in travelers to sub-Saharan Africa. In total, 679 travelers were included in the study. The follow-up rate was 82% (556 of 679). Participants' median travel duration was 12 weeks. There was one incident infection with Strongyloides stercoralis; there were none with Entamoeba histolytica, 4 with Cryptosporidium spp. (1%), and 22 with Giardia lamblia (4%). Nine of 146 travelers (6%) seroconverted for Schistosoma spp. Routine screening of stool samples for parasitic infection is not indicated for asymptomatic people, who travel to the (sub)tropics for up to 3 months. Screening for Schistosoma spp. should be offered to travelers with fresh-water contact in endemic regions.
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.
Assessing the susceptibility of amylose-lysophosphatidylcholine complexes to amylase by the use of iodine
Ahmadi-Abhari, S. ; Woortman, A.J.J. ; Hamer, R.J. ; Loos, K. - \ 2014
Starch-Stärke 66 (2014)5-6. - ISSN 0038-9056 - p. 576 - 581.
wheat-starch - chain-length - inclusion complexes - fatty-acids - in-vivo - digestion - binding - digestibility - microscopy - property
The formation of amylose-lysophosphatidylcholine (LPC) inclusion complexes renders amylose less susceptible to amylase digestion. In order to better understand this phenomenon on a structural level, the complexation of 9% wheat starch suspensions with 0, 2, 3, and 5% exogenous LPC was developed in RVA. Amylose-LPC inclusion complexes were isolated after 15, 30, 60, 120, and 240min in vitro digestion of the wheat starch suspensions to quantify the amount of non-complexed amylose by spectrophotometry. The samples were dissolved in DMSO containing 0.5% LiBr and exposed to iodine. In addition, parts of the digesta were defatted and subjected to the same procedure to elucidate the total amount of amylose that remained undigested. In this way, more insight was obtained into the protective effect of amylose-LPC complex formation on digestion of starch. This study confirms that the amylose susceptibility to amylolysis decreases in the presence of LPC. Higher LPC concentrations not only induced the formation of more amylose inclusion complexes but also resulted in more stable complexes which remained undigested as well as longer amylose chains after enzyme hydrolysis, due to the presence of LPC inside the amylose helix. In addition, a higher melting enthalpy of the amylose-LPC complexes in the digesta demonstrates the protective effect of LPC during enzyme hydrolysis.
An Open Source Image Processing Method to Quantitatively Assess Tissue Growth after Non-Invasive Magnetic Resonance Imaging in Human Bone Marrow Stromal Cell Seeded 3D Polymeric Scaffolds
Leferink, A.M. ; Fratila, R.M. ; Koenrades, M.A. ; Blitterswijk, C.A. van; Velders, A.H. ; Moroni, L. - \ 2014
PLoS One 9 (2014)12. - ISSN 1932-6203
intensity nonuniformity correction - iron-oxide nanoparticles - mesenchymal stem-cells - x-ray microtomography - engineered constructs - articular-cartilage - mri - microscopy - spectroscopy - perfusion
Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional (3D) scaffolds for regenerative medicine and clinical purposes. This is even more important when multipotent human bone marrow stromal cells (hMSCs) are used, as it could offer a method to understand in real time the dynamics of stromal cell differentiation and eventually steer it into the desired lineage. Magnetic Resonance Imaging (MRI) is a promising tool to overcome the challenge of a limited transparency in opaque 3D scaffolds. Technical limitations of MRI involve non-uniform background intensity leading to fluctuating background signals and therewith complicating quantifications on the retrieved images. We present a post-imaging processing sequence that is able to correct for this non-uniform background intensity. To test the processing sequence we investigated the use of MRI for in vitro monitoring of tissue growth in three-dimensional poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) scaffolds. Results showed that MRI, without the need to use contrast agents, is a promising non-invasive tool to quantitatively monitor ECM production and cell distribution during in vitro culture in 3D porous tissue engineered constructs.
IAG ring test feed composition 2014
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Vliege, J.J.M. - \ 2014
Wageningen : RIKILT Wageningen UR (RIKILT report 2014.010) - 21
voersamenstelling - pluimveevoeding - ringtest - microanalyse - microscopie - kippen - feed formulation - poultry feeding - ring test - microanalysis - microscopy - fowls
A ring test was organized for the microscopic determination of composition in animal feed in the framework of the annual ring tests of the IAG – International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The aim of the ring study was to provide the participants information on the performance of the local implementation of the method for composition analysis of feed.
IAG ring test animal proteins 2014
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Voet, H. van der; Vliege, J.J.M. - \ 2014
Wageningen : RIKILT Wageningen UR (RIKILT report 2014.011) - 35
ringtest - microscopie - dierlijke eiwitten - rundveevoeding - pluimveevoeding - diergezondheid - ring test - microscopy - animal proteins - cattle feeding - poultry feeding - animal health
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG – International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method.
Cellphone-based detection platform for rbST biomerker analysis in milk extracts using a microsphere fluorescence immunoassay
Ludwig, S.K.J. ; Zhu, H. ; Phillips, S. ; Shiledar, A. ; Feng, S. ; Tseng, D. ; Ginkel, L.A. van; Nielen, M.W.F. ; Ozcan, A. - \ 2014
Analytical and Bioanalytical Chemistry 406 (2014)27. - ISSN 1618-2642 - p. 6857 - 6866.
recombinant bovine somatotropin - of-care diagnostics - cell-phone - smart-phone - microscopy - cost
Current contaminant and residue monitoring throughout the food chain is based on sampling, transport, administration, and analysis in specialized control laboratories. This is a highly inefficient and costly process since typically more than 99 % of the samples are found to be compliant. On-site simplified prescreening may provide a scenario in which only samples that are suspect are transported and further processed. Such a prescreening can be performed using a small attachment on a cellphone. To this end, a cellphone-based imaging platform for a microsphere fluorescence immunoassay that detects the presence of anti-recombinant bovine somatotropin (rbST) antibodies in milk extracts was developed. RbST administration to cows increases their milk production, but is illegal in the EU and a public health concern in the USA. The cellphone monitors the presence of anti-rbST antibodies (rbST biomarker), which are endogenously produced upon administration of rbST and excreted in milk. The rbST biomarker present in milk extracts was captured by rbST covalently coupled to paramagnetic microspheres and labeled by quantum dot (QD)-coupled detection antibodies. The emitted fluorescence light from these captured QDs was then imaged using the cellphone camera. Additionally, a dark-field image was taken in which all microspheres present were visible. The fluorescence and dark-field microimages were analyzed using a custom-developed Android application running on the same cellphone. With this setup, the microsphere fluorescence immunoassay and cellphone-based detection were successfully applied to milk sample extracts from rbST-treated and untreated cows. An 80 % true-positive rate and 95 % true-negative rate were achieved using this setup. Next, the cellphone-based detection platform was benchmarked against a newly developed planar imaging array alternative and found to be equally performing versus the much more sophisticated alternative. Using cellphone-based on-site analysis in future residue monitoring can limit the number of samples for laboratory analysis already at an early stage. Therewith, the entire monitoring process can become much more efficient and economical.
Nanoscale cell wall deformation impacts long-range bacterial adhesion forces on surfaces
Chen, Y. ; Harapanahalli, A.K. ; Busscher, H.J. ; Norde, W. ; Mei, H.C. van der - \ 2014
Applied and Environmental Microbiology 80 (2014)2. - ISSN 0099-2240 - p. 637 - 643.
staphylococcus-aureus - microscopy - biofilms - attraction - mechanisms - dlvo - van
Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of different short- and long-range forces. Here we present a new atomic force microscopy (AFM)-based method to derive long-range bacterial adhesion forces from the dependence of bacterial adhesion forces on the loading force, as applied during the use of AFM. The long-range adhesion forces of wild-type Staphylococcus aureus parent strains (0.5 and 0.8 nN) amounted to only one-third of these forces measured for their more deformable isogenic ¿pbp4 mutants that were deficient in peptidoglycan cross-linking. The measured long-range Lifshitz-Van der Waals adhesion forces matched those calculated from published Hamaker constants, provided that a 40% ellipsoidal deformation of the bacterial cell wall was assumed for the ¿pbp4 mutants. Direct imaging of adhering staphylococci using the AFM peak force-quantitative nanomechanical property mapping imaging mode confirmed a height reduction due to deformation in the ¿pbp4 mutants of 100 to 200 nm. Across naturally occurring bacterial strains, long-range forces do not vary to the extent observed here for the ¿pbp4 mutants. Importantly, however, extrapolating from the results of this study, it can be concluded that long-range bacterial adhesion forces are determined not only by the composition and structure of the bacterial cell surface but also by a hitherto neglected, small deformation of the bacterial cell wall, facilitating an increase in contact area and, therewith, in adhesion force.
Complex coacervate core micelles as diffusional nanoprobes
Bourouina, N. ; Cohen Stuart, M.A. ; Kleijn, J.M. - \ 2014
Soft Matter 10 (2014). - ISSN 1744-683X - p. 320 - 331.
polymer-solutions - fluorescence recovery - diblock copolymer - probe diffusion - gels - polyelectrolyte - nanoparticles - microscopy - stability - glutaraldehyde
Because of their ease of preparation and versatile modification opportunities, complex coacervate core micelles (C3Ms) may be a good alternative for expensive diffusional probes, such as dendrimers. However, C3Ms are unstable at high salt concentrations and may fall apart in contact with other polymers or (solid) materials. Therefore, we designed and characterized small (15 nm radius), stable fluorescent C3Ms. These were formed by electrostatic interactions between poly(ethylene oxide-methacrylic acid) (PEO–PMAA) and fluorescently labelled poly(allylamine hydrochloride) (PAH) and irreversible cross-linking of the core through amide bonds. We compared the properties of the cross-linked and non-cross-linked micelles. The radii of the two types of micelles were quite similar and independent of the ionic strength. Surprisingly, both were found to be stable at salt concentrations as high as 1.5 M. However, unlike the non-cross-linked C3Ms, the stability of the cross-linked C3Ms is independent of the pH. As a first example of their application as diffusional nanoprobes, we present results on the diffusion of the fluorescent micelles measured in xanthan solutions using fluorescence recovery after photobleaching (FRAP).
IAG ring test animal proteins 2013
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Vliege, J.J.M. - \ 2013
Wageningen : Rikilt - Institute of Food Safety (RIKILT-report 2013.016) - 35
dierlijk eiwit - dierlijke eiwitten - voer - diervoeding - ringtest - microscopie - animal protein - animal proteins - feeds - animal nutrition - ring test - microscopy
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method.
Phosphorescence Imaging of Living Cells with Amino Acid-Functionalized Tris(2-phenylpyridine)iridium(III) Complexes
Steunenberg, P. ; Ruggi, A. ; Berg, N.S. van den; Buckle, T. ; Kuil, J. ; Leeuwen, F.W.B. van; Velders, A.H. - \ 2012
Inorganic Chemistry 51 (2012)4. - ISSN 0020-1669 - p. 2105 - 2114.
cyclometalated iridium complexes - light-emitting-diodes - golgi-apparatus - energy-transfer - emission - ligand - microscopy - ir(iii) - accumulation - derivatives
A series of nine luminescent cyclometalated octahedral iridium(III) tris(2-phenylpyridine) complexes has been synthesized, functionalized with three different amino acids (glycine, alanine, and lysine), on one, two, or all three of the phenylpyridine ligands. All starting complexes and final compounds have been fully analyzed by one-dimensional (ID) and two-dimensional (2D) NMR spectroscopy, and photophysical data have been obtained for all the mono-, bis-, and tri- substituted iridium(III) complexes. Cellular uptake and localization have been studied with flow cytometry and confocal microscopy, respectively. Confocal experiments demonstrate that all nine substituted iridium(III) complexes show variable uptake in the tumor cells. The monosubstituted iridium(III) complexes give the highest cellular uptake, and the series substituted with lysines shows the highest toxicity. This systematic study of amino acid-functionalized Ir(ppy)(3) complexes provides guidelines for further functionalization and possible implementation of luminescent iridium complexes, for example, in (automated) peptide synthesis or biomarker specific targeting.
Development, validation and evaluation of a rapid PCR-nucleic acid lateral flow immuno-assay for the detection of Plasmodium and the differentiation between Plasmodium falciparum and Plasmodium vivax
Mens, P.F. ; Moers, A.P.H.A. ; Bes, L.M. de; Flint, J. ; Sak, J.R.S. ; Keereecharoen, L. ; Overmeir, C. ; Verweij, J.J. ; Hallett, R.L. ; Wihokhoen, B. ; Proux, S. ; Schallig, H.D.F.H. ; Amerongen, A. van - \ 2012
Malaria Journal 11 (2012). - ISSN 1475-2875
malaria diagnosis - amplification - parasite - microscopy - accuracy - tests - care
Background: Molecular tools are very sensitive and specific and could be an alternative for the diagnosis of malaria. The complexity and need for expensive equipment may hamper implementation and, therefore, simplifications to current protocols are warranted. Methods: A PCR detecting the different Plasmodium species and differentiating between Plasmodium falciparum and Plasmodium vivax was developed and combined with a nucleic acid lateral flow immuno-assay (PCR-NALFIA) for amplicon detection. The assay was thoroughly evaluated for the analytical sensitivity and specificity in the laboratory, the robustness and reproducibility in a ring trial and accuracy and predictive value in a field trial. Results: The analytical sensitivity and specificity were 0.978 (95% CI: 0.932-0.994) and 0.980 (95% CI: 0.924-0.997), respectively, and were slightly less sensitive for the detection of P. vivax than for P. falciparum. The reproducibility tested in three laboratories was very good (k = 0.83). This evaluation showed that the PCR machine used could influence the results. Accuracy was evaluated in Thailand and compared to expert microscopy and rapid diagnostic tests (RDTs). The overall and P. falciparum-specific sensitivity and specificity was good ranging from 0.86-1 and 0.95-0.98 respectively, compared to microscopy. Plasmodium vivax detection was better than the sensitivity of RDT, but slightly less than microscopy performed in this study. Conclusion: PCR-NALFIA is a sensitive, specific and robust assay able to identify Plasmodium species with good accuracy. Extensive testing including a ring trial can identify possible bottlenecks before implementation and is therefore essential to perform in additon to other evaluations.
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